Compositions for expanding regulatory t cells (treg), and treating autoimmune and inflammatory diseases and conditions

ABSTRACT

In alternative embodiments, provided are compositions, including e.g., isolated, synthetic or recombinant peptides or polypeptides, for: expanding regulatory T cells (Treg) populations; or, for treating, ameliorating, preventing or reversing a vascular inflammation, and Kawasaki disease (KD) or a pediatric acute vasculitis of the coronary arteries, including vascular coronary abnormalities and the same or similar types of acute or chronic vascular inflammatory abnormalities, and methods for making and using them. In one embodiment, provided are immunotherapies for promoting expansion of natural, regulatory T cells to establish, or re-establish, vascular homeostasis; or, for treating, reversing, preventing or ameliorating: a disease or condition associated with an autoimmune disease or condition; an immune-mediated vascular disorder; a disease or condition that is currently treated with intravenous immunoglobulin (IVIG) therapy; a vascular coronary abnormality; an acute or a chronic vasculitis; an autoimmune inflammatory vasculitis; a T cell mediated pediatric vasculitis; Kawasaki disease (KD) or a pediatric acute vasculitis of the coronary arteries; atherosclerosis; rheumatoid arthritis or Juvenile Idiopathic Arthritis; an autoimmune disease or condition; or, a neoplastic hematological disorder such as a lymphoma (e.g., a T cell lymphoma) or a leukemia.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbers: R01HL103536; UL1 RR031980; and NIH iDASH grant U54Hl108460 by the NIH. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

This invention generally relates to immunology and medicine. Inalternative embodiments, provided are compositions, including e.g.,isolated, synthetic or recombinant peptides, for expanding regulatory Tcells (Treg) populations and treating, ameliorating, preventing orreversing: an autoimmune disease or condition; an acute or chronicvascular inflammation, including Kawasaki disease (KD), a pediatricacute vasculitis of the coronary arteries or a pediatric acutevasculitis of the coronary arteries, and other vascular coronaryabnormalities, including the same or similar types of acute or chronicvascular inflammatory abnormalities, and including an atherosclerosis,and methods for making and using them. In one embodiment, provided areimmunotherapies for promoting expansion of natural, regulatory T cellsto establish, or re-establish, vascular homeostasis.

BACKGROUND OF INVENTION

Kawasaki disease (KD) is an emerging third world pediatric disease thatfrequently leaves lasting coronary abnormalities. Current treatment isintravenous (IV) immunoglobulin (Ig) (or IVIG, or IvIG) infusion, whichis expensive and requires hospitalization and often not an option in thethird world regions where the disease is prevalent.

IVIG therapy generates a specific population of natural regulatory Tcells (nTregs) that recognize the heavy region constant (Fc) ofimmunoglobulin G. Fc-specific Treg are very relevant to maintainvascular homeostasis and can be found in Kawasaki disease subjects thatdo not develop arterial abnormalities after IVIG and in a variety ofacute pediatric febrile controls but not autoimmune diseases. Normalhealthy adults have detectable Fc-specific nTreg in their peripheralblood suggesting that this Treg repertoire is important in controllingvascular homeostasis.

Children that develop coronary arteries abnormalities despite IVIGtreatment do not expand Fc-specific nTreg. Characterization ofFc-specific nTreg clones derived from children that did not developarterial abnormalities after KD suggest that the suppression mechanismof pro-inflammatory T cell responses (by Fc-specific nTreg) occur in thelymph nodes, and Fc-specific nTregs are activated by resident B cells.IgG+ mature B cells can activate Fc-specific nTreg in the absence ofsoluble Fc fragments. The characterization of immunodominant Fc peptidesthat bind multiple Human Leukocytes Antigens (HLA) alleles would be agreat alternative to IVIG.

SUMMARY OF THE INVENTION

In alternative embodiments, provided are isolated, synthetic orrecombinant peptides having a sequence comprising or consisting of:

(SEQ ID NO: 1) TAALGCLVKDYFPEP (SEQ ID NO: 2) CLVKDYFPEPVTVSW (SEQ IDNO: 3) SVFLFPPKPKDTLMI (SEQ ID NO: 4) PPKPKDTLMISRTPE (SEQ ID NO: 5)KLTVDKSRWQQGNVF (SEQ ID NO: 6) KSRWQQGNVFSCSVM (SEQ ID NO: 7)NGQPENNYKTTPPVL (SEQ ID NO: 8) NNYKTTPPVLDSDGS (SEQ ID NO: 9)TFPAVLQSSGLYSLS (SEQ ID NO: 10) LQSSGLYSLSSVVTV (SEQ ID NO: 11)LYSLSSVVTVPSSSL (SEQ ID NO: 12) SVVTVPSSSLGTQTY (SEQ ID NO: 13)EQYNSTYRVVSVLTV, (SEQ ID NO: 14) TYRVVSVLTVLHQDW, (SEQ ID NO: 15)TPPVLDSDGSFFLYS, (SEQ ID NO: 16) QGNVFSCSVMHEALH, or (SEQ ID NO: 17)SCSVMHEALHNHYTQ,

wherein in alternative embodiments provided are methods and uses foradministering at least one, two, three, four, five, six, seven or eightor more or all of the isolated, synthetic or recombinant peptides to anindividual, and optionally this administration generates naturalregulatory T cells (nTregs) that can suppress pro-inflammatory T cellsand pro-inflammatory T cell responses, and optionally the individual isa human.

In alternative embodiments, the isolated, synthetic or recombinantpeptide as provided herein has at least one conservative amino acidsubstitution and retains its property of generating (ability togenerate) natural regulatory T cells (nTregs) that can suppresspro-inflammatory T cells and pro-inflammatory T cell responses whenadministered to an individual,

wherein optionally the at least one conservative amino acid substitutioncomprises substituting an amino acid with another amino acid of likecharacteristics; or, a conservative substitution comprises: replacementof an aliphatic amino acid with another aliphatic amino acid;replacement of a Serine with a Threonine or vice versa; replacement ofan acidic residue with another acidic residue; replacement of a residuebearing an amide group with another residue bearing an amide group;exchange of a basic residue with another basic residue; or replacementof an aromatic residue with another aromatic residue.

In alternative embodiments, the peptide, or at least one amino acid inthe peptide, comprises or consists of a peptidomimetic or a non-naturalamino acid residue.

In alternative embodiments, provided herein are isolated, synthetic orrecombinant nucleic acids comprising or consisting of a sequenceencoding a peptide or polypeptide as provided herein, wherein optionallythe nucleic acid further comprises, or is contained within, anexpression cassette, a plasmid, an expression vector or a recombinantvirus, wherein optionally the nucleic acid, or the expression cassette,plasmid, expression vector or recombinant virus is contained with a cell(optionally a “host cell”), optionally a human cell or a non-human cell,and optionally the cell is transformed with the nucleic acid, or theexpression cassette, plasmid, expression vector or recombinant virus. Inalternative embodiments, provided herein are host cells transduced,transfected or otherwise engineered to contain within an isolated,synthetic or recombinant peptide or polypeptide as provided hereinand/or an isolated, synthetic or recombinant nucleic acid or anexpression cassette, plasmid, expression vector or recombinant virus asprovided herein.

In alternative embodiments, provided are compositions comprising: oneisolated, synthetic or recombinant peptide, or two, three, four, five,six, seven, eight or more or all isolated, synthetic or recombinantpeptides as provided herein; or, an isolated, synthetic or recombinantnucleic acid, or an expression cassette, a plasmid, an expression vectoror a recombinant virus, or a cell, as provided herein.

In alternative embodiments, provided are pharmaceutical compositions orformulations comprising: one isolated, synthetic or recombinant peptide,or two, three, four or five, six, seven, eight, nine or ten or moreisolated, synthetic or recombinant peptides as provided herein; anisolated, synthetic or recombinant nucleic acid, or an expressioncassette, a plasmid, an expression vector or a recombinant virus, or acell, as provided herein; or, a composition as provided herein, and apharmaceutically acceptable excipient. In alternative embodiments, thepharmaceutical composition or formulation is formulated foradministration via a parenteral route comprising or consisting of asubcutaneous, an intravenous (IV), an intradermal, an intramuscular, anintraperitoneal, an intranasal, a transdermal or a buccal route; or thepharmaceutical composition or formulation is formulated for oral ortopical administration; or the pharmaceutical composition or formulationis formulated for administration intradermally as a sterile formulation,or as an inhaled powder, or is formulated for administration as acontrolled release formulation, a tablet, a pill, a gel, a patch, in animplant or in a spray. In alternative embodiments, the pharmaceuticalcomposition or formulation is formulated as a sterile solution forinjection, or as a powder or a lyophilized (freeze-dried) composition.In alternative embodiments, the pharmaceutical composition orformulation is formulated as a liquid, an emulsion, a lyophilizedpowder, a spray, a cream, a lotion, a controlled release formulation, atablet, a pill, a gel, a patch, in an implant or in a spray; or isformulated as an aqueous or a non-aqueous isotonic sterile injectionsolution, or an aqueous or a non-aqueous sterile suspension.

In alternative embodiments, provided are liposomes comprising: anisolated, synthetic or recombinant peptide as provided herein; anisolated, synthetic or recombinant nucleic acid, or an expressioncassette, a plasmid, an expression vector or a recombinant virus, or acell, as provided herein; a composition as provided herein; apharmaceutical or formulation as provided herein; or, any combinationthereof.

In alternative embodiments, provided are nanoparticles comprising: anisolated, synthetic or recombinant peptide as provided herein; anisolated, synthetic or recombinant nucleic acid, or an expressioncassette, a plasmid, an expression vector or a recombinant virus, or acell, as provided herein; a composition as provided herein; apharmaceutical or formulation as provided herein; a liposome as providedherein; or, any combination thereof.

In alternative embodiments, provided are therapeutic combinationscomprising two, three, four, five, six, seven, eight or more or all ofthe isolated, synthetic or recombinant peptides as provided herein; anisolated, synthetic or recombinant nucleic acid, or an expressioncassette, a plasmid, an expression vector or a recombinant virus, or acell, as provided herein; a composition as provided herein; apharmaceutical or formulation as provided herein; a liposome as providedherein; or, nanoparticles as provided herein,

wherein optionally the therapeutic combination is used for:

(a) immune-regulation by expanding natural regulatory T cells (nTregs)or nTreg suppression of pro-inflammatory T cells and/or pro-inflammatoryT cell responses; or

(b) the treatment, prevention or amelioration of:

-   -   a disease or condition associated with an immune-mediated        vascular disorder;    -   a disease or condition that is currently treated with        intravenous immunoglobulin (IVIG) therapy;    -   a disease or condition ameliorated by immune regulation        comprising suppression of pro-inflammatory T cells and/or        suppression of pro-inflammatory T cell responses;    -   sa vascular coronary abnormality;    -   an autoimmune inflammatory vasculitis;    -   an acute or a chronic vasculitis (or vascular inflammation);    -   a T cell mediated pediatric vasculitis;    -   Kawasaki disease (KD) or a pediatric acute vasculitis of the        coronary arteries;    -   an atherosclerosis;    -   rheumatoid arthritis;    -   Juvenile Idiopathic Arthritis;    -   an autoimmune disease or condition; or    -   a neoplastic hematological disorder, a leukemia or a lymphoma,        such as a T cell lymphoma.

In alternative embodiments, provided are vaccines comprising: anisolated, synthetic or recombinant peptide as provided herein; anisolated, synthetic or recombinant nucleic acid, or an expressioncassette, a plasmid, an expression vector or a recombinant virus, or acell, as provided herein; a composition as provided herein; apharmaceutical or formulation as provided herein; a liposome as providedherein, a nanoparticle as provided herein; a therapeutic combination asprovided herein; or, any combination thereof, for the manufacture of amedicament or a vaccine,

wherein optionally the vaccine is administered with an adjuvant or anincomplete adjuvant.

In alternative embodiments, provided are Uses of: an isolated, syntheticor recombinant peptide as provided herein; an isolated, synthetic orrecombinant nucleic acid, or an expression cassette, a plasmid, anexpression vector or a recombinant virus, or a cell, as provided herein;a composition as provided herein; a pharmaceutical or formulation asprovided herein; a liposome as provided herein, a nanoparticle asprovided herein; a therapeutic combination as provided herein; a vaccineas provided herein; or, any combination thereof, for the manufacture ofa medicament or a vaccine.

In alternative embodiments, provided are uses of: an isolated, syntheticor recombinant peptide as provided herein; an isolated, synthetic orrecombinant nucleic acid, or an expression cassette, a plasmid, anexpression vector or a recombinant virus, or a cell, as provided herein;a composition as provided herein; a pharmaceutical or formulation asprovided herein; a liposome as provided herein, a nanoparticle asprovided herein; a therapeutic combination as provided herein; a vaccineas provided herein; or, any combination thereof, for the manufacture ofa medicament or a vaccine for:

(a) immune-regulation by expanding natural regulatory T cells (nTregs)or nTreg suppression of pro-inflammatory T cells and/or pro-inflammatoryT cell responses; or

(b) the treatment, prevention or amelioration of:

-   -   a disease or condition associated with an immune-mediated        vascular disorder;    -   a disease or condition that is currently treated with        intravenous immunoglobulin (IVIG) therapy;    -   a disease or condition ameliorated by immune regulation        comprising suppression of pro-inflammatory T cells and/or        suppression of pro-inflammatory T cell responses;    -   sa vascular coronary abnormality;    -   an autoimmune inflammatory vasculitis;    -   an acute or a chronic vasculitis (or vascular inflammation);    -   a T cell mediated pediatric vasculitis;    -   Kawasaki disease (KD) or a pediatric acute vasculitis of the        coronary arteries;    -   an atherosclerosis;    -   rheumatoid arthritis,    -   Juvenile Idiopathic Arthritis,    -   an autoimmune disease or condition; or    -   a neoplastic hematological disorder, a leukemia or a lymphoma,        such as a T cell lymphoma.

In alternative embodiments, provided are methods for:

(a) effecting immune-regulation or immune homeostasis by expandingnatural regulatory T cells (nTregs) or causing or promoting nTregsuppression (partial or complete) of pro-inflammatory T cells and/orpro-inflammatory T cell responses; or

(b) the treatment, prevention or amelioration of:

-   -   a disease or condition associated with an immune-mediated        vascular disorder;    -   a disease or condition that is currently treated with        intravenous immunoglobulin (IVIG) therapy;    -   a disease or condition ameliorated by immune regulation        comprising suppression of pro-inflammatory T cells and/or        suppression of pro-inflammatory T cell responses;    -   a vascular coronary abnormality;    -   an acute or a chronic vasculitis (or vascular inflammation);    -   an autoimmune inflammatory vasculitis;    -   a T cell mediated pediatric vasculitis;    -   Kawasaki disease (KD) or a pediatric acute vasculitis of the        coronary arteries;    -   an atherosclerosis;    -   rheumatoid arthritis,    -   Juvenile Idiopathic Arthritis,    -   an autoimmune disease or condition; or    -   a neoplastic hematological disorder, a leukemia or a lymphoma,        such as a T cell lymphoma;

comprising:

administering to an individual in need thereof an effective amount of:an isolated, synthetic or recombinant peptide as provided herein; anisolated, synthetic or recombinant nucleic acid, or an expressioncassette, a plasmid, an expression vector or a recombinant virus, or acell, as provided herein; a composition as provided herein; apharmaceutical or formulation as provided herein; a liposome as providedherein, a nanoparticle as provided herein; a therapeutic combination asprovided herein; a vaccine as provided herein; or, any combinationthereof, to an individual in need thereof,

thereby:

effecting immune-regulation or immune homeostasis by expanding naturalregulatory T cells (nTregs) or causing or promoting nTreg suppression ofpro-inflammatory T cells and/or pro-inflammatory T cell responses; or

treating, preventing or ameliorating:

-   -   a disease or condition associated with an immune-mediated        vascular disorder;    -   a disease or condition that is currently treated with        intravenous immunoglobulin (IVIG) therapy;    -   a disease or condition ameliorated by immune regulation        comprising suppression of pro-inflammatory T cells and/or        suppression of pro-inflammatory T cell responses;    -   a vascular coronary abnormality;    -   an acute or a chronic vasculitis (or vascular inflammation);    -   an autoimmune inflammatory vasculitis;    -   a T cell mediated pediatric vasculitis;    -   Kawasaki disease (KD) a pediatric acute vasculitis of the        coronary arteries;    -   an atherosclerosis;    -   rheumatoid arthritis,    -   Juvenile Idiopathic Arthritis,    -   an autoimmune disease or condition; or    -   a neoplastic hematological disorder, a leukemia or a lymphoma,        such as a T cell lymphoma.

In alternative embodiments of methods as provided herein, an isolated,synthetic or recombinant peptide or polypeptide as provided herein; anisolated, synthetic or recombinant nucleic acid, or an expressioncassette, a plasmid, an expression vector or a recombinant virus, or acell, as provided herein; a composition as provided herein; apharmaceutical or formulation as provided herein; a liposome as providedherein, a nanoparticle as provided herein; a therapeutic combination asprovided herein; a vaccine as provided herein; or, any combinationthereof, is administered:

-   -   via an enteral or a parenteral route, or orally, or systemically        or topically;    -   via a parenteral route comprising a subcutaneous, an intravenous        (IV), an intradermal, an intramuscular, an intraperitoneal, an        intranasal, a transdermal or a buccal route;    -   intradermally as a sterile formulation;    -   as a controlled release formulation, a tablet, a pill, a gel, a        patch, in an implant or in a spray, as an inhaled powder; or    -   with a non-specific immuno-stimulator, wherein optionally the        non-specific immuno-stimulator comprises or consists of a        granulocyte-macrophage colony-stimulating factor polypeptide; or        sargramostim, or LEUKINE™ (Bayer, Leverkusen, Germany).

In alternative embodiments of methods as provided herein, an isolated,synthetic or recombinant peptide or polypeptide as provided herein; anisolated, synthetic or recombinant nucleic acid, or an expressioncassette, a plasmid, an expression vector or a recombinant virus, or acell, as provided herein; a composition as provided herein; apharmaceutical or formulation as provided herein; a liposome as providedherein, a nanoparticle as provided herein; a therapeutic combination asprovided herein; a vaccine as provided herein; or, any combinationthereof, is administered:

-   -   parenterally by one bolus injection, or by two, three or four or        more bolus injections, or by gradual perfusion over time;    -   at intervals of 1 week, 2 weeks, 4 weeks (or one month), 6        weeks, 8 weeks (or two months) or one year; or    -   at a daily dose of peptide in a range of about 10 nanograms to        10 milligrams, or about 1 microgram to 10 milligrams.

In alternative embodiments of methods as provided herein,

an isolated, synthetic or recombinant peptide as provided herein; anisolated, synthetic or recombinant nucleic acid, or an expressioncassette, a plasmid, an expression vector or a recombinant virus, or acell, as provided herein; a composition as provided herein; apharmaceutical or formulation as provided herein; a liposome as providedherein, a nanoparticle as provided herein; a therapeutic combination asprovided herein; a vaccine as provided herein; or, any combinationthereof, is administered:

as a prophylactic or preventative measure to an individual having apersonal history of, or to an individual having a detected genotype orphenotype indicating a predisposition to:

-   -   a disease or condition associated with an immune-mediated        vascular disorder;    -   a disease or condition that is currently treated with        intravenous immunoglobulin (IVIG) therapy;    -   a disease or condition ameliorated by immune regulation        comprising suppression of pro-inflammatory T cells and/or        suppression of pro-inflammatory T cell responses;    -   a vascular coronary abnormality;    -   an acute or a chronic vasculitis (or vascular inflammation);    -   an autoimmune inflammatory vasculitis;    -   a T cell mediated pediatric vasculitis;    -   Kawasaki disease (KD) or a pediatric acute vasculitis of the        coronary arteries;    -   an atherosclerosis;    -   rheumatoid arthritis,    -   Juvenile Idiopathic Arthritis,    -   an autoimmune disease or condition; or    -   a neoplastic hematological disorder, a leukemia or a lymphoma,        such as a T cell lymphoma.

The details of one or more embodiments as provided herein are set forthin the description below. Other features, objects, and advantagesexemplary embodiments as provided herein will be apparent from thedescription and from the claims.

All publications, patents, patent applications, GenBank sequences andATCC deposits, cited herein are hereby expressly incorporated byreference for all purposes.

The embodiments of the description described herein are not intended tobe exhaustive or to limit the disclosure to the precise forms disclosedin the following drawings or detailed description. Rather, theembodiments are chosen and described so that others skilled in the artcan appreciate and understand the principles and practices of thedescription.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings set forth herein are illustrative of embodiments of theinvention and are not meant to limit the scope of the invention asencompassed by the claims.

FIG. 1A and FIG. 1B graphically illustrates: IL-10 secretion bypeptide-specific Treg; Treg lines from the KD patient with coronaryartery aneurysm (CAA) are shown as KD 3873, right-hand-most graphiccolumn in “KD Sub-Acute” side of the figure; FIG. 1B graphicallyillustrates a representative enumeration of a cell sorting scan of CD4+CD25^(high) T cells in response to the whole Fc and peptide pool121-135; 126-140 in PBMC cultures from a CAA+ KD subject with aneurysms;as described in detail in Example 1, below.

FIG. 2 graphically illustrates data of ELISA assays of supernatantsharvested and tested for IL-10, IFN-gamma and IL-2 secretion from: PBMCderived from sub-acute KD subjects which secrete IL-10, but notIFN-gamma or IL-2 in response to scalar doses of Fc, PBMC from 15 KDsubjects (10 with normal arteries after IVIG therapy and 5 with dilatedarteries or aneurysm after IVIG therapy) cultured with scalar doses ofpurified Fc fragments for 4 days in the absence of exogenouslymphokines, as described in detail in Example 2, below.

FIG. 3A and FIG. 3B graphically illustrates percent CD4+ CD25^(high)Treg cell expansion to scalar doses of Fc (as μg/ml) as analyzed by flowcytometry in PBMC cultures derived from 15 sub-acute KD subjects, withnormal arteries and 5 with dilated arteries or aneurysm previouslystudied for lymphokine production, PBMC were cultured for 4 days with 0(no antigen), 1, 10, or 100<g/ml of purified Fc fragments; FIG. 3B(which is FIG. 2B, in Example 2) graphically illustrates a summary ofthe Treg response in 15 sub-acute KD subjects described in FIG. 3A asthe percent increase Fc-specific Treg in Sub-acute KD subjects afterIVIG, and normal coronary arteries, as described in detail in Example 2,below.

FIG. 4 graphically illustrates data showing the differential expansionof Fc-specific Treg from the acute to the sub-acute phase of KDdepending upon clinical outcome, where the the fold-increase of theFc-specific Treg repertoire from the acute to sub-acute phase in 6 KDsubjects was studied: three subjects with normal coronary arteries afterIVIG therapy, three subjects with dilated arteries or aneurysm despiteIVIG therapy, as described in detail in Example 2, below.

FIG. 5 graphically illustrates cell sorting data showing FOXP3+ T cellsexpansion in response to Fc: intracellular staining for FOXP3^(high)expression in CD4+ T cells was assessed in Fc-stimulated PBMC culturesfrom two subacute patients after IVIG therapy, where “Subject 15” haddilated coronary arteries by echocardiogram; and “Subject 16” had normalcoronary arteries and showed expansion of the Treg population inresponse to 100<g/ml Fc, as described in detail in Example 2, below.

FIG. 6 graphically illustrates data showing activated CD4+ and CD8+CD25^(low) DR^(high) but not memory IL15r+ T cells decrease in PBMCcultured with Fc when Treg expand: PBMC from two sub-acute KD subjectswith normal arteries after IVIG (Subject 18 is upper graphicillustration and Subject 19 is lower graphic illustration) were culturedwith 100<g/ml purified Fc fragments or media alone as control to addressthe fate of activated T cells and memory T cells in Fc-stimulatedcultures in which Treg expand. Treg were defined as CD4+ CD25^(high);activated CD4+ and CD8+ T cells as CD25^(low) and DR^(high); and memoryT cells as IL-15r+, as described in detail in Example 2, below.

FIG. 7A and FIG. 7B graphically illustrate data showing characterizationof Fc-specific Treg clones: 3 representative Treg clones were generatedfrom a KD subject 17 two weeks following IVIG treatment: FIG. 7A: Leftgraphic illustration: Production of IL-10, IL-4 and TGFβ measured byELISA 48 hours after stimulation with autologous, irradiated B cellspulsed with 20 μg/ml Fc fragments; FIG. 7A Right graphic illustration:qRT-PCR analysis of cell lysates from the same three Treg clones; and,FIG. 7B illustrates phenotype of a representative Treg clonecharacterized as CD4+ CD25^(high), intracellular FOXP3^(high),CD45RA^(low), IL-7r^(low), IL15r−, CCR6−, CCR7^(high) and CCR4−, asdescribed in detail in Example 2, below.

FIG. 8A and FIG. 8B illustrates data showing that Fc-specific Tregclones recognize endogenous processed IgG presented by autologous Bcells in an MHC-restricted, TcR-mediated manner: FIG. 8A: Live, but notparaformaldehyde-fixed, autologous EBV-transformed IgG+|B cellsstimulate Treg clones to secrete IL-10 and IL-4 in the absence ofexogenous Fc; FIG. 8B: only autologous, but not allogeneic IgG+ B cellsactivate a Fc-specific Treg clone, as described in detail in Example 2,below.

FIG. 9 graphically illustrates data showing that Treg (the characterizedas CD4+CD25^(high) T cells) expand in response to Fc in acute pediatricinflammatory conditions but not in subjects with acute autoimmuneconditions, where Fc-induced Treg expansion in cultured PBMC wasdetected after Fc stimulation in 7 subjects with acute viral infections,2 subjects with bacterial infections and 1 subject with a systemic drugreaction, as described in detail in Example 2, below.

FIG. 10A and FIG. 10B graphically illustrate cell sorting data showingTreg response to purified Fc and F(ab)2 fragments in sub-acute KDsubjects: FIG. 10A: CD4+ CD25^(high) Treg expansion in PBMC culturesfrom sub-acute KD subjects with normal arteries after IVIG therapy inresponse to scalar doses of Fc; and, FIG. 10B: lack of CD4+ CD25^(high)Treg expansion in response to F(ab)2 fragments in 4 patients within thesame cohort, as described in detail in Example 2, below.

FIG. 11 graphically illustrates the Fc-specific clonal Treg response isTcR mediated: two representative Fc-specific Treg clones secrete IL-10when stimulated 24 hours in vitro with an anti-CD3 agonistic antibody,as described in detail in Example 2, below.

FIG. 12 illustrates Table 1 of Example 3, which summarizes thedemographic and clinical status of pediatric KD study subjects, asdescribed in detail in Example 3, below.

FIG. 13 illustrates Table 3 of Example 3, which summarizes the total 64peptides, each 15 amino acids in length, with a 10 amino acid-overlapfor each peptide, spanning the whole Fc molecule, that were used todefine the fine specificity of Fc-specific nTreg; the table illustratesthe amino acid sequences of the 15-mer overlapping peptides, asdescribed in detail in Example 3, below.

FIG. 14, or FIG. 1 of Example 3, graphically illustrates data showingnTreg fine specificities in sub-acute and convalescent KD subjects,where 2×10⁵ PBMC/well derived from 10 sub-acute and 6 convalescent KDsubjects were cultured with pools of two Fc peptides (Table 3, see FIG.13) for 4 days in the absence of exogenous lymphokines and IL-10secretion (in pg/ml) in response to peptide stimulation was measured inculture supernatants by ELISA on day 4, as described in detail inExample 3, below.

FIG. 15, or FIG. 2 of Example 3, graphically illustrates cell sortingdata showing the response of nTreg to peptide pool 13; enumeration ofCD4+ CD25^(high) T cells from KD subject #10 in response to Fc 121-135and 126-140 (pool 13) is illustrated, and the most immunogenic sequencesin this cohort of patients (P7, upper right), as described in detail inExample 3, below.

FIG. 16, or FIG. 3 of Example 3, graphically illustrates data showingthat IL-10 secretion in PBMC cultures from healthy adult donors inresponse to peptide pools: 2×10⁵ PBMC/well derived from six healthyadult donors were cultured with pools of two Fc peptides (Table 3, seeFIG. 13) for 4 days in the absence of exogenous lymphokines, and IL-10secretion by nTreg in response to peptide stimulation served as a readout in these experiments and was measured in culture supernatants byELISA on day 4, as described in detail in Example 3, below.

FIG. 17A and FIG. 17B, or FIG. 4 of Example 3, graphically illustratedata showing that pools 3 and 4 are more immunogenic in healthy donorsthan in KD patients: FIG. 17A: IL-10 secretion in response to amino acidresidues 21-35 and 26-40 (pool 3) and 31-45 and 36-50 (pool 4) inhealthy donors; FIG. 17B: nTreg responses to pools 3 and 4 in KDpatients: only 2 of 12 KD subjects responded to these peptide pools, asdescribed in detail in Example 3, below.

FIG. 18A and FIG. 18B, or FIG. 5 of Example 3, graphically illustratedata showing CD4+ CD25^(high) nTreg expansion in response to scalardoses of Fc; PBMC were cultured for 4 days with 0, 1, 10, or 100 μg/mlpurified Fc fragments: FIG. 18A: Fc-specific nTreg response in adultsubjects who had KD in childhood; FIG. 18B: Fc-specific nTreg responsein healthy adult controls, as described in detail in Example 3, below.

FIG. 19, or FIG. 6 of Example 3, graphically illustrates data showingHLA binding predictions of peptides derived from the Fc sequence; IEDBconsensus algorithm was used to predict HLA class II binding affinity ofthe Fc sequences described in Table 3, see FIG. 13; and immunogenicpeptide pools are indicated, as described in detail in Example 3, below.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

In alternative embodiments, provided are peptides and polypeptides,formulations and pharmaceutical compositions, and methods of using them,to generate immunogenic effects that help balance, or re-balance, the Tcell repertoire in patients with, e.g., Kawasaki Disease and relatedvascular inflammations and cardiac conditions. In alternativeembodiments, the immunogenic effect is to promote expansion of natural,regulatory T cells (nTregs) to establish, or re-establish, vascularhomeostasis. In alternative embodiments, compositions and methods asprovided herein are used as a substitute for, or as a complement to,intravenous (IV) immunoglobulin (Ig) (or IVIG, or IvIG) therapy,particularly for Kawasaki disease (KD), but also more generally for thesame or similar types of acute or chronic vascular inflammatoryabnormalities. In alternative embodiments, compositions and methods asprovided herein are used for treating, preventing or ameliorating: adisease or condition associated with an immune-mediated vasculardisorder; a disease or condition that is currently treated withintravenous immunoglobulin (IVIG) therapy; a vascular coronaryabnormality; an acute or a chronic vasculitis; an autoimmuneinflammatory vasculitis; a T cell-mediated pediatric vasculitis;Kawasaki disease (KD); atherosclerosis; rheumatoid arthritis or JuvenileIdiopathic Arthritis; or, a neoplastic hematological disorder such as alymphoma (e.g., a T cell lymphoma) or a leukemia.

The Fc protein (heavy region constant of IgG) is not recognized by nTregin a fraction of patients, but the peptides as provided herein canrescue the nTreg response. While the invention is not limited by anyparticular mechanism of action, peptides and polypeptides as providedherein can overcome the problem of defective antigen processing and HLAbinding that jeopardize nTreg responses to the Fc protein by directlystimulating anti-inflammatory nTregs. Thus, in alternative embodiments,compositions and methods provided herein are used as an “optimized”alternative to IVIG based on the immunodominant Fc peptides providedherein. Peptides and polypeptide provided herein can be stable, low costand easy to administer.

In alternative embodiments, peptides and polypeptide provided herein areused (e.g., are administered) in incomplete adjuvants, e.g., analogousto the formulation of peptides that are in Phase III for theimmunotherapy of a variety of tumors, as a peptide-based approach toinduce immune-regulation.

In alternative embodiments, compositions as provided herein, includingpeptides and and polypeptide provided herein, formulations andpharmaceutical compositions, are used (e.g., are administered) as apeptide-based immunotherapy to raise immune-regulation via nTregexpansion, e.g., to treat, prevent, reverse, or ameliorate vasculardiseases like, e.g., Kawasaki disease, or any disease or condition thatis currently treated with IVIG or that is responsive to (e.g., isameliorated or treated by) immune-regulation comprising suppression ofpro-inflammatory T cells and/or suppression of pro-inflammatory T cellresponses. In alternative embodiments, compositions as provided herein,including peptides, formulations and pharmaceutical compositions, areused (e.g., are administered) to treat, prevent, reverse or ameliorateany immune-mediated vascular disorder, e.g., atherosclerosis; rheumatoidarthritis or Juvenile Idiopathic Arthritis; or, a neoplastichematological disorder such as a lymphoma (e.g., a T cell lymphoma) or aleukemia. While the invention is not limited by any particular mechanismof action, patients with Juvenile Idiopathic Arthritis lack Fc-specificnTreg, and including peptides, formulations and pharmaceuticalcompositions (e.g., immunodominant Fc peptides as provided herein) canby-pass lack of specific T cell-mediated regulation in a variety ofclinical settings.

Intravenous immunoglobulin therapy (IVIG) is the treatment of choice formany immune-mediated diseases, yet its mechanisms of action areincompletely elucidated. We investigated the possibility that IVIGplayed a direct role in the expansion of regulatory T cells (Treg) thatrecognize the heavy chain constant region of immunoglobulin G (Fc) as amechanism for the recovery of Kawasaki disease or a pediatric acutevasculitis of the coronary arteries, e.g., a T cell mediated pediatricvasculitis of the coronary arteries. We successfully generatedFc-specific Treg clones from sub-acute KD subjects that did not developarterial complications after IVIG and defined an unusual functionalphenotype: Fc-specific Treg secrete IL-10 and small amounts of IL-4 butnot TGF-β. Antigen presentation studies demonstrated that these Tregclones can be activated by autologous B cells that express IgG on theircell surface in the absence of exogenous Fc. The IgG molecule has to becanonically processed and presented by autologous MHC molecules to berecognized by Treg. In support of the importance of this novel Tregpopulation in downsizing vascular inflammation, KD patients with dilatedcoronary arteries or aneurysms despite IVIG treatment failed to expandFc-specific Treg. Our results point to a specificity of a previouslyun-described Treg population for the clinical benefit provided by IVIGtherapy in children.

Peptides and Polypeptides

In alternative embodiments, peptides or polypeptides as provided hereinor peptides or polypeptides used to practice methods or uses as providedherein comprise a recombinant protein, a synthetic protein, apeptidomimetic, a non-natural peptide, or a combination thereof.Peptides and proteins used to practice compositions, methods and uses asprovided herein can be recombinantly expressed in vitro or in vivo. Thepeptides and polypeptides as provided herein can be made and isolatedusing any method known in the art as well as using the methods describedherein. Polypeptide and peptides provided herein or for practicingmethods and uses as provided herein can also be synthesized, whole or inpart, using chemical methods well known in the art. See e.g., Caruthers(1980) Nucleic Acids Res. Symp. Ser. 215-223; Horn (1980) Nucleic AcidsRes. Symp. Ser. 225-232; Banga, A. K., Therapeutic Peptides andProteins, Formulation, Processing and Delivery Systems (1995) TechnomicPublishing Co., Lancaster, Pa. For example, peptide synthesis can beperformed using various solid-phase techniques (see e.g., Roberge (1995)Science 269:202; Merrifield (1997) Methods Enzymol. 289:3-13) includingany automated polypeptide synthesis process known in the art.

In alternative embodiments, peptides as provided herein or peptides usedto practice methods or uses as provided herein are synthetic moleculesincluding, e.g., peptidomimetics and non-natural amino acids. Inalternative aspects, peptides and polypeptide provided herein compriseamino acids joined to each other by peptide bonds or modified peptidebonds and may comprise modified amino acids other than the 20gene-encoded amino acids.

In alternative embodiments, peptides as provided herein or peptides usedto practice methods or uses as provided herein have many types ofmodifications, e.g., modifications including glycosylation, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of a phosphatidylinositol,cross-linking cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristolyation, oxidation, pegylation, phosphorylation, prenylation,racemization, selenoylation, sulfation and transfer-RNA mediatedaddition of amino acids to protein such as arginylation. See forexample, Creighton, T. E., Proteins—Structure and Molecular Properties2nd Ed., W.H. Freeman and Company, NY (1993); Posttranslational CovalentModification of Proteins, B. C. Johnson, Ed., Academic Press, NY, pp.1-12 (1983)). In another embodiment, a DRP can be glycol-pegylated asdescribed in U.S. Pat. No. 7,405,198; or can be glycosylated asdescribed in U.S. Pat. No. 7,276,475 or 7,399,613, or 7,338,933, thelater describing O-linked glycosylation of peptides. Peptides asprovided herein or peptides used to practice methods or uses as providedherein can be acylated as described e.g., in U.S. Pat. No. 7,273,921.

In alternative embodiments, peptides as provided herein or peptides usedto practice methods or uses as provided herein can comprise any“mimetic” and/or “peptidomimetic” form. In alternative embodiments,peptides as provided herein or peptides used to practice methods or usesas provided herein comprise synthetic chemical compounds which havesubstantially the same structural and/or functional characteristics ofnatural or non-natural peptides. A mimetic provided herein can be eitherentirely composed of synthetic, non-natural analogues of amino acids,or, is a chimeric molecule of partly natural peptide amino acids andpartly non-natural analogs of amino acids. A mimetic used to practicecompositions, methods or uses as provided herein can also incorporateany amount of natural or non-natural amino acid conservativesubstitutions as long as such substitutions also do not substantiallyalter the mimetic's structure and/or activity.

Routine experimentation will determine whether a synthetic molecule ormimetic is effective for practicing a composition, method or use asprovided herein, e.g., for effecting immune-regulation by expandingnatural regulatory T cells (nTregs) or nTreg suppression ofpro-inflammatory T cells and/or pro-inflammatory T cell responses.Methodologies detailed herein and others known to persons skilled in theart may be used to select or guide one to choose effective mimetic forpracticing the compositions and/or methods as provided herein.

Polypeptide mimetic compositions for practicing compositions, methods oruses as provided herein can comprise any combination of non-naturalstructural components. In alternative aspects, mimetic compositions forpracticing compositions, methods or uses as provided herein can compriseone or all of the following three structural groups: a) residue linkagegroups other than the natural amide bond (“peptide bond”) linkages; b)non-natural residues in place of naturally occurring amino acidresidues; or c) residues which induce secondary structural mimicry,i.e., to induce or stabilize a secondary structure, e.g., a beta turn,gamma turn, beta sheet, alpha helix conformation, and the like. Forexample, a polypeptide can be characterized as a mimetic when all orsome of its residues are joined by chemical means other than naturalpeptide bonds. Individual peptidomimetic residues can be joined bypeptide bonds, other chemical bonds or coupling means, such as, e.g.,glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides,N,N′-dicyclohexylcarbodiimide (DCC) or N,N′-diisopropylcarbodiimide(DIC). Linking groups that can be an alternative to the traditionalamide bond (“peptide bond”) linkages include, e.g., ketomethylene (e.g.,—C(═O)—CH₂— for —C(═O)—NH—), aminomethylene (CH₂—NH), ethylene, olefin(CH═CH), ether (CH₂—O), thioether (CH₂—S), tetrazole (CN₄—), thiazole,retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistryand Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp267-357, “Peptide Backbone Modifications,” Marcell Dekker, NY). Apolypeptide can also be characterized as a mimetic by containing all orsome non-natural residues in place of naturally occurring amino acidresidues. Non-natural residues are well described in the scientific andpatent literature; a few exemplary non-natural compositions useful asmimetics of natural amino acid residues and guidelines are describedbelow. Mimetics of aromatic amino acids can be generated by replacingby, e.g., D- or L-naphylalanine; D- or L-phenylglycine; D- or L-2thieneylalanine; D- or L-1, -2, 3-, or 4-pyreneylalanine; D- or L-3thieneylalanine; D- or L-(2-pyridinyl)-alanine; D- orL-(3-pyridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- orL-(4-isopropyl)-phenylglycine; D-(trifluoromethyl)-phenylglycine;D-(trifluoromethyl)-phenylalanine; D-p-fluoro-phenylalanine; D- orL-p-biphenylphenylalanine; D- or L-p-methoxy-biphenylphenylalanine; D-or L-2-indole(alkyl)alanines; and, D- or L-alkylainines, where alkyl canbe substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl,pentyl, isopropyl, iso-butyl, sec-isotyl, iso-pentyl, or a non-acidicamino acids. Aromatic rings of a non-natural amino acid include, e.g.,thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl,pyrrolyl, and pyridyl aromatic rings.

Mimetics of acidic amino acids used to practice methods or uses asprovided herein can be generated by substitution by, e.g.,non-carboxylate amino acids while maintaining a negative charge;(phosphono)alanine; sulfated threonine. Carboxyl side groups (e.g.,aspartyl or glutamyl) can also be selectively modified by reaction withcarbodiimides (R′—N—C—N—R′) such as, e.g.,1-cyclohexyl-3(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide. Aspartyl orglutamyl can also be converted to asparaginyl and glutaminyl residues byreaction with ammonium ions. Mimetics of basic amino acids can begenerated by substitution with, e.g., (in addition to lysine andarginine) the amino acids ornithine, citrulline, or (guanidino)-aceticacid, or (guanidino)alkyl-acetic acid, where alkyl is defined above.Nitrile derivative (e.g., containing the CN-moiety in place of COOH) canbe substituted for asparagine or glutamine. Asparaginyl and glutaminylresidues can be deaminated to the corresponding aspartyl or glutamylresidues. Arginine residue mimetics can be generated by reacting arginylwith, e.g., one or more conventional reagents, including, e.g.,phenylglyoxal, 2,3-butanedione, 1,2-cyclo-hexanedione, or ninhydrin,e.g., under alkaline conditions. Tyrosine residue mimetics can begenerated by reacting tyrosyl with, e.g., aromatic diazonium compoundsor tetranitromethane. N-acetylimidizol and tetranitromethane can be usedto form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.Cysteine residue mimetics can be generated by reacting cysteinylresidues with, e.g., alpha-haloacetates such as 2-chloroacetic acid orchloroacetamide and corresponding amines; to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteine residue mimetics can also begenerated by reacting cysteinyl residues with, e.g.,bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic acid;chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide;methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4nitrophenol; or, chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimeticscan be generated (and amino terminal residues can be altered) byreacting lysinyl with, e.g., succinic or other carboxylic acidanhydrides. Lysine and other alpha-amino-containing residue mimetics canalso be generated by reaction with imidoesters, such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,trinitro-benzenesulfonic acid, O-methylisourea, 2,4, pentanedione, andtransamidase-catalyzed reactions with glyoxylate. Mimetics of methioninecan be generated by reaction with, e.g., methionine sulfoxide. Mimeticsof proline include, e.g., pipecolic acid, thiazolidine carboxylic acid,3- or 4-hydroxy proline, dehydroproline, 3- or 4-methylproline, or3,3,-dimethylproline. Histidine residue mimetics can be generated byreacting histidyl with, e.g., diethylprocarbonate or para-bromophenacylbromide. Other mimetics that can be used include, e.g., those generatedby hydroxylation of proline and lysine; phosphorylation of the hydroxylgroups of seryl or threonyl residues; methylation of the alpha-aminogroups of lysine, arginine and histidine; acetylation of the N-terminalamine; methylation of main chain amide residues or substitution withN-methyl amino acids; or amidation of C-terminal carboxyl groups.

Peptides used to practice methods or uses as provided herein cancomprise tags or signal sequences, i.e., leader sequences, e.g., foridentifying or secreting a peptide or a polypeptide provided herein froma production host cell. In one embodiment, a cleavable linker is placedbetween the signal sequence or tag and the peptide or a polypeptideprovided herein.

Generating and Manipulating Nucleic Acids

In alternative aspects, because the peptides as provided herein orpeptides used to practice methods or uses as provided herein can be usedin recombinant form, also provided are nucleic acids, which themselvescan be recombinant, to make them. In alternative embodiments, nucleicacids s provided herein or nucleic acids used to practice methods oruses as provided herein are made, isolated and/or manipulated by, e.g.,cloning and expression of cDNA libraries, amplification of message orgenomic DNA by PCR, and the like.

The nucleic acids provided herein or nucleic acids used to practicemethods or uses as provided herein, whether RNA, cDNA, genomic DNA,vectors, viruses or hybrids thereof, can be isolated from a variety ofsources, genetically engineered, amplified, and/or expressed/generatedrecombinantly. Recombinant polypeptides (e.g., a peptide used topractice a method or use as provided herein) generated from thesenucleic acids can be individually isolated or cloned and tested for adesired activity. Any recombinant expression system can be used,including e.g. in vitro, bacterial, fungal, mammalian, yeast, insect orplant cell expression systems.

In one embodiment, nucleic acids s provided herein or nucleic acids usedto practice methods or uses as provided herein are synthesized in vitroby well-known chemical synthesis techniques, as described in, e.g.,Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic AcidsRes. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380;Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol.68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett.22:1859; U.S. Pat. No. 4,458,066.

Techniques for the manipulation of nucleic acids s provided herein ornucleic acids used to practice methods or uses as provided herein, suchas, e.g., subcloning, labeling probes (e.g., random-primer labelingusing Klenow polymerase, nick translation, amplification), sequencing,hybridization and the like are well described in the scientific andpatent literature, see, e.g., Sambrook, ed., MOLECULAR CLONING: ALABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring Harbor Laboratory,(1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley& Sons, Inc., New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY ANDMOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I.Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).

Also provided are fusion proteins and nucleic acids encoding them, anduses thereof. Any peptide used to practice methods and uses as providedherein can be fused to a heterologous peptide or polypeptide. Inalternative embodiments, a heterologous peptide or polypeptide joined orfused to a protein used to practice methods and uses as provided hereincan be an N-terminal identification peptide which imparts a desiredcharacteristic, such as fluorescent detection, increased stabilityand/or simplified purification.

Peptides used to practice methods and uses as provided herein can alsobe synthesized and expressed as fusion proteins with one or moreadditional domains linked thereto for, e.g., producing a moreimmunogenic peptide, to more readily isolate a recombinantly synthesizedpeptide, to identify and isolate antibodies and antibody-expressing Bcells, and the like. Detection and purification facilitating domainsinclude, e.g., metal chelating peptides such as polyhistidine tracts andhistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp, Seattle Wash.). The inclusion of acleavable linker sequences such as Factor Xa or enterokinase(Invitrogen, San Diego Calif.) between a purification domain and themotif-comprising peptide or polypeptide to facilitate purification. Forexample, an expression vector can include an epitope-encoding nucleicacid sequence linked to six histidine residues followed by a thioredoxinand an enterokinase cleavage site (see e.g., Williams (1995)Biochemistry 34:1787-1797; Dobeli (1998) Protein Expr. Purif.12:404-414). The histidine residues facilitate detection andpurification while the enterokinase cleavage site provides a means forpurifying the epitope from the remainder of the fusion protein.Technology pertaining to vectors encoding fusion proteins andapplication of fusion proteins are well described in the scientific andpatent literature, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53.

Pharmaceutical Compositions and Formulations

Provided are pharmaceutical compositions and formulations comprisingpeptides and polypeptide as provided herein for treating, preventing,reversing or ameliorating: a disease or condition associated with animmune-mediated vascular disorder; a disease or condition that iscurrently treated with intravenous immunoglobulin (IVIG) therapy; avascular coronary abnormality; an acute or a chronic vasculitis; anautoimmune inflammatory vasculitis; a T cell mediated pediatricvasculitis; Kawasaki disease or a pediatric acute vasculitis of thecoronary arteries; atherosclerosis; rheumatoid arthritis or JuvenileIdiopathic Arthritis; an immune disease or condition; or, a neoplastichematological disorder such as a lymphoma (e.g., a T cell lymphoma) or aleukemia.

In alternative embodiments, the compositions as provided herein areformulated with a pharmaceutically acceptable carrier. In alternativeembodiments, the pharmaceutical compositions and formulations asprovided herein can be administered parenterally, topically, orally orby local administration, such as by aerosol or transdermally. Thepharmaceutical compositions can be formulated in any way and can beadministered in a variety of unit dosage forms depending upon thecondition or disease (e.g., type of acute or chronic vasculitides) andthe degree of illness, the general medical condition of each patient,the resulting preferred method of administration and the like. Detailson techniques for formulation and administration of pharmaceuticals andpeptides are well described in the scientific and patent literature,see, e.g., the latest edition of Remington's Pharmaceutical Sciences,Maack Publishing Co, Easton Pa. (“Remington's”).

Peptides as provided herein can be administered alone or as a componentof a pharmaceutical formulation (composition). The compounds may beformulated for administration, e.g., as a peptide, in any convenient wayfor use in human or veterinary medicine. Wetting agents, incompleteadjuvants, emulsifiers and lubricants, such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, release agents, coatingagents, sweetening, flavoring and perfuming agents, preservatives andantioxidants can also be present in the compositions e.g., peptideformulations.

Formulations or compositions as provided herein, e.g., peptideformulations, include those suitable for intradermal, inhalation,oral/nasal, topical, parenteral, rectal, and/or intravaginaladministration. The formulations may conveniently be presented in unitdosage form and may be prepared by any methods well known in the art ofpharmacy. The amount of active ingredient (e.g., a peptide as providedherein) which can be combined with a carrier material to produce asingle dosage form will vary depending upon the host being treated, theparticular mode of administration, e.g., intradermal or inhalation. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the compound which produces a therapeutic effect, e.g., an antigenspecific T cell or humoral response.

Pharmaceutical formulations as provided herein, e.g., peptideformulations, can be prepared according to any method known to the artfor the manufacture of pharmaceuticals or peptides. Such drugs cancontain sweetening agents, flavoring agents, coloring agents andpreserving agents. A formulation can be admixtured with nontoxicpharmaceutically acceptable excipients which are suitable formanufacture. Formulations may comprise one or more diluents,emulsifiers, preservatives, buffers, excipients, etc. and may beprovided in such forms as liquids, powders, emulsions, lyophilizedpowders, sprays, creams, lotions, controlled release formulations,tablets, pills, gels, on patches, in implants, etc.

Pharmaceutical formulations for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art inappropriate and suitable dosages. Such carriers enable thepharmaceuticals to be formulated in unit dosage forms as tablets, pills,powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries,suspensions, etc., suitable for ingestion by the patient. Pharmaceuticalpreparations for oral use can be formulated as a solid excipient,optionally grinding a resulting mixture, and processing the mixture ofgranules, after adding suitable additional compounds, if desired, toobtain tablets or dragee cores. Suitable solid excipients arecarbohydrate or protein fillers include, e.g., sugars, includinglactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice,potato, or other plants; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxy-methylcellulose; andgums including arabic and tragacanth; and proteins, e.g., gelatin andcollagen. Disintegrating or solubilizing agents may be added, such asthe cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate. Push-fit capsules can contain activeagents mixed with a filler or binders such as lactose or starches,lubricants such as talc or magnesium stearate, and, optionally,stabilizers. In soft capsules, the active agents can be dissolved orsuspended in suitable liquids, such as fatty oils, liquid paraffin, orliquid polyethylene glycol with or without stabilizers.

Aqueous suspensions, e.g., peptide formulations, can contain an activeagent (e.g., a peptide or peptidomimetic as provided herein) inadmixture with excipients suitable for the manufacture of aqueoussuspensions, e.g., for aqueous intradermal injections. Such excipientsinclude a suspending agent, such as sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose, sodium alginate,polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing orwetting agents such as a naturally occurring phosphatide (e.g.,lecithin), a condensation product of an alkylene oxide with a fatty acid(e.g., polyoxyethylene stearate), a condensation product of ethyleneoxide with a long chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a partialester derived from a fatty acid and a hexitol (e.g., polyoxyethylenesorbitol mono-oleate), or a condensation product of ethylene oxide witha partial ester derived from fatty acid and a hexitol anhydride (e.g.,polyoxyethylene sorbitan mono-oleate). The aqueous suspension can alsocontain one or more preservatives such as ethyl or n-propylp-hydroxybenzoate, one or more coloring agents, one or more flavoringagents and one or more sweetening agents, such as sucrose, aspartame orsaccharin. Formulations can be adjusted for osmolarity.

In one embodiment, oil-based pharmaceuticals are used for administrationof hydrophobic peptides as provided herein. Oil-based suspensions can beformulated by suspending an active agent in a vegetable oil, such asarachis oil, olive oil, sesame oil or coconut oil, or in a mineral oilsuch as liquid paraffin; or a mixture of these. See e.g., U.S. Pat. No.5,716,928 describing using essential oils or essential oil componentsfor increasing bioavailability and reducing inter- and intra-individualvariability of orally administered hydrophobic pharmaceutical compounds(see also U.S. Pat. No. 5,858,401). The oil suspensions can contain athickening agent, such as beeswax, hard paraffin or cetyl alcohol.Sweetening agents can be added to provide a palatable oral preparation,such as glycerol, sorbitol or sucrose. These formulations can bepreserved by the addition of an antioxidant such as ascorbic acid. As anexample of an injectable oil vehicle, see Minto (1997) J. Pharmacol.Exp. Ther. 281:93-102.

Pharmaceutical formulations, e.g., peptide formulations, as providedherein can also be in the form of oil-in-water emulsions. The oily phasecan be a vegetable oil or a mineral oil, described above, or a mixtureof these. Suitable emulsifying agents include naturally-occurring gums,such as gum acacia and gum tragacanth, naturally occurring phosphatides,such as soybean lecithin, esters or partial esters derived from fattyacids and hexitol anhydrides, such as sorbitan mono-oleate, andcondensation products of these partial esters with ethylene oxide, suchas polyoxyethylene sorbitan mono-oleate. The emulsion can also containsweetening agents and flavoring agents, as in the formulation of syrupsand elixirs. Such formulations can also contain a demulcent, apreservative, or a coloring agent. In one embodiment, peptides asprovided herein are formulated as oil-in-water emulsions as described inU.S. Pat. No. 7,371,395, describing an injectable oil-in-water emulsion,comprising an aqueous solution containing at least one peptides asprovided herein, a mineral oil, a non-ionic lipophilic surfactant and/ora non-ionic hydrophilic surfactant having a high hydrophilic-lipophilicbalance (HLB) value. In alternative embodiments, these injectableoil-in-water emulsions as provided herein comprise a paraffin oil, asorbitan monooleate, an ethoxylated sorbitan monooleate and/or anethoxylated sorbitan trioleate.

In practicing methods and uses as provided herein, the pharmaceuticalcompounds, e.g., peptide formulations, can also be administered by inintranasal, intraocular and intravaginal routes including suppositories,insufflation, powders and aerosol formulations (for examples of steroidinhalants, see e.g., Rohatagi (1995) J. Clin. Pharmacol. 35:1187-1193;Tjwa (1995) Ann. Allergy Asthma Immunol. 75:107-111). Suppositoriesformulations can be prepared by mixing the drug with a suitablenon-irritating excipient which is solid at ordinary temperatures butliquid at body temperatures and will therefore melt in the body torelease the drug. Such materials are cocoa butter and polyethyleneglycols.

In practicing methods and uses as provided herein, the pharmaceuticalcompounds, e.g., peptide formulations, can be delivered bytransdermally, by a topical route, formulated as applicator sticks,solutions, suspensions, emulsions, gels, creams, ointments, pastes,jellies, paints, powders, and aerosols.

In practicing methods and uses as provided herein, the pharmaceuticalcompounds, e.g., peptide formulations, can also be delivered asmicrospheres for slow release in the body. For example, microspheres canbe administered via intradermal injection of drug which slowly releasesubcutaneously; see Rao (1995) J. Biomater Sci. Polym. Ed. 7:623-645; asbiodegradable and injectable gel formulations, see, e.g., Gao (1995)Pharm. Res. 12:857-863 (1995); or, as microspheres for oraladministration, see, e.g., Eyles (1997) J. Pharm. Pharmacol. 49:669-674.

In practicing methods and uses as provided herein, the pharmaceuticalcompounds, e.g., peptide formulations, can be parenterally administered,such as by intravenous (IV) administration or administration into a bodycavity or lumen of an organ. These formulations can comprise a solutionof active agent dissolved in a pharmaceutically acceptable carrier.Acceptable vehicles and solvents that can be employed are water andRinger's solution, an isotonic sodium chloride. In addition, sterilefixed oils can be employed as a solvent or suspending medium. For thispurpose any bland fixed oil can be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid can likewisebe used in the preparation of injectables. These solutions are sterileand generally free of undesirable matter. These formulations may besterilized by conventional, well known sterilization techniques. Theformulations may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents, e.g.,sodium acetate, sodium chloride, potassium chloride, calcium chloride,sodium lactate and the like. The concentration of active agent in theseformulations can vary widely, and will be selected primarily based onfluid volumes, viscosities, body weight, and the like, in accordancewith the particular mode of administration selected and the patient'sneeds. For IV administration, the formulation can be a sterileinjectable preparation, such as a sterile injectable aqueous oroleaginous suspension. This suspension can be formulated using thosesuitable dispersing or wetting agents and suspending agents. The sterileinjectable preparation can also be a suspension in a nontoxicparenterally-acceptable diluent or solvent, such as a solution of1,3-butanediol. The administration can be by bolus or continuousinfusion (e.g., substantially uninterrupted introduction into a bloodvessel for a specified period of time).

The pharmaceutical compounds and formulations, e.g., peptideformulations, as provided herein can be lyophilized. Provided are astable lyophilized formulation comprising a composition as providedherein, which can be made by lyophilizing a solution comprising apharmaceutical as provided herein and a bulking agent, e.g., mannitol,trehalose, raffinose, and sucrose or mixtures thereof. A process forpreparing a stable lyophilized formulation can include lyophilizing asolution about 2.5 mg/mL protein, about 15 mg/mL sucrose, about 19 mg/mLNaCl, and a sodium citrate buffer having a pH greater than 5.5 but lessthan 6.5. See, e.g., U.S. patent app. no. 20040028670.

The compositions and formulations, e.g., peptide formulations, asprovided herein can be delivered by the use of liposomes. By usingliposomes, particularly where the liposome surface carries ligandsspecific for target cells, or are otherwise preferentially directed to aspecific organ, one can focus the delivery of the active agent intotarget cells in vivo. See, e.g., U.S. Pat. Nos. 6,063,400; 6,007,839;Al-Muhammed (1996) J. Microencapsul. 13:293-306; Chonn (1995) Curr.Opin. Biotechnol. 6:698-708; Ostro (1989) Am. J. Hosp. Pharm.46:1576-1587.

The formulations and pharmaceuticals as provided herein can beadministered for prophylactic and/or therapeutic treatments. Inalternative embodiments, for therapeutic applications, compositions areadministered to a subject already suffering from a condition; this canbe called a “therapeutically effective amount”. For example, inalternative embodiments, pharmaceutical compositions as provided hereinare administered in an amount sufficient to generate immune-regulationby expanding natural regulatory T cells (nTregs) or nTreg suppression ofpro-inflammatory T cells and/or pro-inflammatory T cell responses. Theamount of pharmaceutical composition adequate to accomplish this isdefined as a “therapeutically effective dose.” The dosage schedule andamounts effective for this use, i.e., the “dosing regimen,” will dependupon a variety of factors, including the stage of the disease (e.g., adisease or condition associated with an immune-mediated vasculardisorder) or condition, the severity of the disease or condition, thegeneral state of the patient's health, the patient's physical status,age and the like. In calculating the dosage regimen (e.g., peptideadministration regimen) for a patient, the mode of administration alsois taken into consideration, e.g., intradermal injection of peptideformulation.

The dosage regimen also takes into consideration pharmacokineticsparameters well known in the art, i.e., the active agents' rate ofabsorption, bioavailability, metabolism, clearance, and the like (see,e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617;Groning (1996) Pharmazie 51:337-341; Fotherby (1996) Contraception54:59-69; Johnson (1995) J. Pharm. Sci. 84:1144-1146; Rohatagi (1995)Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24:103-108;the latest Remington's, supra). The state of the art allows theclinician to determine the dosage regimen for each individual patient,active agent and disease or condition treated. Guidelines provided forsimilar compositions used as pharmaceuticals can be used as guidance todetermine the dosage regiment, i.e., dose schedule and dosage levels,administered practicing the methods are uses as provided herein arecorrect and appropriate.

Single or multiple administrations of formulations, e.g., peptideformulations, can be given depending on for example: the dosage andfrequency as required and tolerated by the patient, the degree andamount of antigen specific CTLs and/or T helper cells, orantigen-specific antibodies, generated after each peptideadministration, and the like. The formulations should provide asufficient quantity of active agent to effectively treat, prevent orameliorate a conditions, diseases or symptoms, e.g., generateimmune-regulation by expanding natural regulatory T cells (nTregs) ornTreg suppression of pro-inflammatory T cells and/or pro-inflammatory Tcell responses.

In alternative embodiments, pharmaceutical formulations, e.g., peptideformulations, for oral administration are in a daily amount of betweenabout 0.1 to 0.5 to about 20, 50, 100 or 1000 or more ug per kilogram ofbody weight per day. In an alternative embodiment, dosages are fromabout 1 mg to about 4 mg per kg of body weight per patient per day areused. Lower dosages can be used, in contrast to administration orally,into the blood stream, into a body cavity or into a lumen of an organ.Substantially higher dosages can be used in topical or oraladministration or administering by powders, spray or inhalation. Actualmethods for preparing parenterally or non-parenterally administrableformulations will be known or apparent to those skilled in the art andare described in more detail in such publications as Remington's, supra.

The methods and uses as provided herein can further compriseco-administration with other drugs or pharmaceuticals, e.g.,compositions for treating cancer, septic shock, infection, fever, painand related symptoms or conditions. For example, the methods, usesand/or compositions and formulations as provided herein can beco-administered with antibiotics (e.g., antibacterial or bacteriostaticpeptides or proteins), particularly those effective against gramnegative bacteria, fluids, cytokines, immunoregulatory agents,anti-inflammatory agents, complement activating agents, such as peptidesor proteins comprising collagen-like domains or fibrinogen-like domains(e.g., a ficolin), carbohydrate-binding domains, and the like andcombinations thereof.

Formulations

Provided are polypeptide and peptide formulations and methods for makingand using them. In alternative embodiments, peptides as provided hereinare formulated as aqueous solutions and administered intradermally aspeptides.

In alternative embodiments, the peptide is conjugated or linked toanother peptide or administered with another protein, such as a carrierprotein or immunogenic protein. Such factors are known in the art and itis well within the skill of physicians and immunologists to make suchdeterminations.

The peptide active agent can be present in a formulation as providedherein in varying concentrations, e.g., in one embodiment the minimumconcentration of peptide is an amount necessary to generateimmune-regulation by expanding natural regulatory T cells (nTregs) ornTreg suppression of pro-inflammatory T cells and/or pro-inflammatory Tcell responses, while the maximum concentration is the maximum amountthat will remain in solution or homogeneously suspended within theinitial mixture. In alternative embodiments, the minimum concentrationof peptide is an amount necessary to generate immune-regulation byexpanding natural regulatory T cells (nTregs) or nTreg suppression ofpro-inflammatory T cells and/or pro-inflammatory T cell responses, andthe maximum concentration is the point at which a homogeneous suspensioncannot be maintained. In alternative embodiments, doses can comprise 1to 100 μg of protein antigen, or 5 to 50 μg, or 5 to 25 μg. A desiredamount of peptide varies from formulation to formulation, or applicationto application (e.g., form of vasculitis) but is easily determinable byone of skill in the art. Peptide preparation is well known in the art,see e.g., Peptide Design (“The subunit and adjuvant approach” Eds.Powell M. F. & Newman M. J. (1995) Plenum Press New York).

Methods of delivering the peptide are also well known in the art. Forexample, in alternative embodiments peptides as provided herein areformulated and delivered via a parenteral route comprising or consistingof a subcutaneous, an intravenous (IV), an intradermal, anintramuscular, an intraperitoneal, an intranasal, a transdermal or abuccal route.

In alternative embodiments peptides as provided herein are deliveredintradermally or intra-epidermally using any needle-like structures ordevice, e.g., as described in U.S. Patent App. Pub. No. 20090012494,describing use of microneedle devices, e.g., with rows of hollowmicroneedles. In alternative embodiments peptides as provided herein aredelivered using micro-cannula, e.g., as described in U.S. Pat. No.7,473,247. When using this or another device or needle to practicemethods and uses as provided herein, peptide formulations can bedirectly targeted into an intradermal space; or can be delivered into anintradermal space as a bolus or by infusion. In alternative embodiments,“intradermal” is administration of a peptide formulation as providedherein into the dermis in such a manner that the peptide as providedherein therein readily reaches the richly vascularized papillary dermiswhere it can be rapidly systemically absorbed, or the peptide can betaken up directly by cells (e.g., dendritic cells) in the skin. Inalternative embodiments, “intradermal” includes every layer of the skin,including stratum corneum, epidermis and dermis.

In one embodiment, a drug-delivery patch is used to deliver a peptideformulation as provided herein, e.g., as described in U.S. Patent App.Pub. No. 20090010998. In one embodiment, provided is a drug-deliverypatch having at least one dissolvable layer comprising a peptide asprovided herein and an adhesive backing or cover. In one embodiment, anindividual is transdermally vaccinated by ablating an area of thestratum corneum of the individual and applying the patch to that area.

Methods for determining the efficacy of a peptide formulation asprovided herein, or a particular administration of a peptide formulationas provided herein, are well known in the art. For example, cell-basedor humoral responses can be assessed (measured) using in vitro basedassays and/or in vivo based assays, including animal based assays.Assays for measuring cell-based or humoral immune response are wellknown in the art, e.g., see, Coligan et al., (eds.), 1997, CurrentProtocols in Immunology, John Wiley and Sons, Inc. Cell-based or humoralimmune responses may be detected and/or quantitated using standardmethods known in the art including, e.g., an ELISA assay, chromiumrelease assays and the like. The humoral immune response may be measuredby detecting and/or quantitating the relative amount of an antibodywhich specifically recognizes an antigenic or immunogenic agent in thesera of a subject who has been treated with a peptide formulation asprovided herein relative to the amount of the antibody in an untreatedsubject. ELISA assays can be used to determine total antibody titers ina sample obtained from a subject treated with an agent as providedherein.

Kits and Packages

Provided are kits, packets and packages comprising compositions andcells (e.g., dendritic cells) as provided herein and, in some aspects,instructions for practicing methods and uses as provided herein,including the peptide formulations or drug-delivery patches as providedherein. In alternative embodiments, storage devices, such as vials, andpeptide delivery devices such as drug-delivery patches comprisingpeptide formulations as provided herein are provided herein.

These and many other variations and embodiments as provided herein willbe apparent to one of skill in the art upon a review of the appendeddescription and examples. The invention will be further described withreference to the following examples; however, it is to be understoodthat the invention is not limited to such examples.

EXAMPLES Example 1: Peptides Effective for Expanding Regulatory T Cells(Treg) Populations

This example presents data demonstrating that compositions as providedherein effectively expand regulatory T cells (Treg) populations for,e.g., ameliorating vascular inflammation, and Kawasaki disease or apediatric acute vasculitis of the coronary arteries, including vascularcoronary abnormalities and the same or similar types of acute or chronicvascular inflammatory abnormalities. Peptides as provided herein can berecognized by nTreg without further antigen processing.

Fc-specific nTregs can suppress pro-inflammatory T cells andpro-inflammatory T cell responses. To define the fine specificity ofnTreg after IVIG for therapeutic purposes, we developed a panel ofpeptides 15 amino acid long, overlapping 10 amino acids, that cover thewhole (entire) Fc sequences.

We successfully identified in vitro a panel of unique Fc sequences neverreported before that generate nTreg not only in KD patients that do notdevelop arterial complications after IVIG but also in patients thatdevelop arterial complication and do not respond to the whole Fc proteinby expanding specific nTreg:

1. Fc sequence 21-35 (SEQ ID NO: 1) TAALGCLVKDYFPEP 2. Fc sequence 26-40(SEQ ID NO: 2) CLVKDYFPEPVTVSW 3. Fc sequence 121-135 (SEQ ID NO: 3)SVFLFPPKPKDTLMI 4. Fc sequence 126-140 (SEQ ID NO: 4) PPKPKDTLMISRTPE 5.Fc sequence 291-305 (SEQ ID NO: 5) KLTVDKSRWQQGNVF 6. Fc sequence296-310 (SEQ ID NO: 6) KSRWQQGNVFSCSVM 7. Fc sequence 266-280 (SEQ IDNO: 7) NGQPENNYKTTPPVL 8. Fc sequence 271-285 (SEQ ID NO: 8)NNYKTTPPVLDSDGS Fc sequence 276-290 (SEQ ID NO: 15) TPPVLDSDGSFFLYS Fcsequence 301-315 (SEQ ID NO: 16) QGNVFSCSVMHEALH Fc sequence 306-320(SEQ ID NO: 17) SCSVMHEALHNHYTQ

Within peptides 9 to 12 some amino acids are included within theTregitopes previously published:

9. Fc sequence 51-65 (SEQ ID NO: 9) TFPAVLQSSGLYSLS (sequence includedin Tregitope 167) 10. Fc sequence 56-70 (SEQ ID NO: 10) LQSSGLYSLSSVVTV(sequence included in Tregitope 167) 5 11. Fc sequence 61-75 (SEQ ID NO:11) LYSLSSVVTVPSSSL (sequence included in Tregitope 167) 12. Fc sequence66-80 (SEQ ID NO: 12) SVVTVPSSSLGTQTY (sequence included in Tregitope167) 10 13. Fc sequence 176-190 (SEQ ID NO: 13) EQYNSTYRVVSVLTV(sequence included in Tregitope 289) 14. Fc sequence 181-195 (SEQ ID NO:14) TYRVVSVLTVLHQDW (sequence included in Tregitope 289).

These exemplary immunodominant Fc peptides as provided herein have beenidentified in three cohorts of subjects:

1) Kawasaki disease sub-acute patients 2 weeks after IVIG;2) Patients that had Kawasaki disease 1-2 year earlier treated withIVIG;3) Healthy donors that never received IVIG.

These unique sequences are recognized by nTreg in the large majority ofthe patients studied after IVIG, KD patients treated with IVIG 1 to 2years earlier and normal healthy controls that did not receive IVIG.

Kawasaki Disease (KD) patients with arterial complication do not respondto the whole Fc protein but they do respond to 15mers Fc peptides asprovided herein, as illustrated in FIG. 1. Notably, the immunodominantFc peptides recognized by nTreg in KD patients after IVIG are the sameas the one recognized by nTreg in normal healthy donors, suggesting thatthe sequences identified are universal.

FIG. 1 of Example 1 illustrates data from Fc peptide epitope mapping in3 healthy adult donors and 3 Kawasaki disease (KD) patients witharterial complication who do not respond to the whole Fc protein but theKD patients do respond to 15mers Fc peptides as provided herein afterIVIG. PBMC were cultured with Fc peptides (as described above) for 4days in the absence of lymphokines. FIG. 1A graphically illustrates:IL-10 secretion by peptide-specific Treg; Treg lines from the KD patientwith coronary artery aneurysm (CAA) are shown as KD 3873,right-hand-most graphic column in “KD Sub-Acute” side of the figure.FIG. 1B graphically illustrates a representative enumeration of a cellsorting scan of CD4+ CD25^(high) T cells in response to the whole Fc andpeptide pool 121-135; 126-140 in PBMC cultures from a CAA+KD subjectwith aneurysms.

Example 2: Treg Populations Effective for Resolving VascularInflammation

This example describes a novel Treg population that specificallyrecognizes the Fc of IgG presented by autologous mature IgG+ Blymphocytes, that expands following IVIG infusion, and that is distinctfrom the previously described Treg that recognize exogenous pan-DR Fcepitopes in normal healthy donors (24). Expansion of this Fc-specificTreg population is associated with resolution of vascular inflammation,while failure to expand is associated with progressive damage to thevascular wall and coronary artery aneurysm formation in infants andyoung children with KD.

Materials and Methods Study Population

KD patients and pediatric patients with acute inflammatory conditionswere enrolled at Rady Children's Hospital San Diego following parentalinformed consent. The protocol was approved by the Institutional ReviewBoard at UCSD. All KD patients enrolled in this study (Table 1) wereevaluated by echocardiography during the acute admission and at two andsix weeks following diagnosis.

TABLE 1 KD subjects enrolled in the study. Coronary artery Fc-specificKD subject # Age, yrs Sex Race/Ethnicity Illness day^(a) Z-max^(b) Tregresponse  1 1.4 M Hispanic 4 0.8 +  2 5.4 F Caucasian/Asian 5 0.7 +  30.6 M Hispanic/Asian 6 1.4 +  4 2.0 M Caucasian/Hispanic/ 8 1.7 + NativeAmerican  5 5.8 F Hispanic 5 1.1 +  6 5.3 M Caucasian 5 1.2 +  7 0.3 MAsian 3 1.2 +  8 3.7 M Caucasian 8 0.9 +  9 6.9 M Caucasian 6 1.0 + 102.9 M Hispanic 7 1.6 + 11 1.9 M Caucasian 4 3.3 (dilated) − 12 7.9 MHispanic 4 2.8 (dilated) − 13 2.0 F Mixed 8 4.7 (aneurysm) − 14 0.9 MHispanic 6 5.6 (aneurysm) − 15 5.7 F Asian 10 2.7 (dilated) − 16 1.6 MCaucasian 5 1.3 + 17 2.9 F Hispanic 5 −0.1   + 18 6.4 F Asian 6 1.0 + 191.9 M Asian/Caucasian 8 2.3 + 20 0.8 M Hispanic 4 8.1 (aneurysm) − 215.6 M Asian/Caucasian 4 1.5 + Subjects 6, 16, 20 were also treated withinfliximab. ^(a)Illness day 1 = onset of fever. ^(b)Z-max scores =maximum internal diameter for the left anterior descending or rightcoronary artery expressed as standard deviation units from the mean(Z-score) normalized for body surface area.

The internal diameter of the right and left anterior descending coronaryarteries was measured and expressed as Z scores (standard deviationunits from the mean normalized for body surface area; normal Zscore<2.5). Z_(worst) was defined as the highest Z score of eithercoronary artery measured during the first six weeks after fever onset.All KD subjects were treated with IVIG 2 gram (g)/kilogram (kg) andaspirin 80 to 100 mg/kd/day until afebrile, then 3 to 5 mg/kg day untilthe platelet count had returned to normal. All subjects were taking lowdose aspirin at the time of the sub-acute phlebotomy. No subject in thisseries had IVIG resistance defined as persistent or recrudescent feverat least 36 h after completion of the initial WIG infusion. Subjects 6,16, and 20 received infliximab 5 mg/kg IV prior to IVIG infusion as partof a clinical trial of intensification of initial WIG therapy(clinicaltrials.gov).

Heparinized blood samples (1-4 ml) were obtained prior to IVIG treatmentat the time of diagnosis (acute) and two weeks after IVIG therapy(sub-acute). Pediatric patients with acute inflammatory conditionsstudied as controls (acute viral infections, bacterial infections,systemic drug allergy, juvenile idiopathic arthritis, andHenoch-Schoenlein purpura) had blood sampled only once and were aged1.6-15.4 yrs. (Table 2):

TABLE 2 Acute pediatric febrile controls. Control Age, Illness subject #yrs Sex Race/Ethnicity day Final diagnosis 1 2.3 F Hispanic 14 Viralstomatitis 2 2.1 M Hispanic ~3-5 Viral syndrome 3 1.6 F Hispanic 5 Viralsyndrome 4 6.6 F Hispanic/ 6 Viral Caucasian 5 1.7 F American 4Viral/her angina Indian 6 5.8 F Caucasian 6 Adenovirus 7 9.6 M Hispanic10 Viral syndrome 8 3.6 F Caucasian 2 Abscess 9 2.2 M Asian 5 Viral 1015.4 F Caucasian 2 Bactrim drug reaction 11 2.3 F Caucasian 16 Juvenileidiopathic arthritis (JIA) 12 3.8 F Hispanic 10 Juvenile idiopathicarthritis (JIA) 13 4.6 F Hispanic ~21 Henoch-Schoenlein purpura (HSP)

Fc-Specific Treg Detection and Characterization of Treg Phenotype

To enumerate Fc-specific Treg that activate after IVIG infusion, wedeveloped a method to avoid non-specific expansion of Treg bytolerogenic DC or the expansion of effector T cells. PBMC were platedwith scalar doses of purified Fc (1, 10 and 100 μg/ml; Life MeridianScience) at a concentration of 4×10⁵ cells/well in 96 flat-bottomedplates (Falcon) for 4 days. Cell cultures did not receive any exogenousIL-2 prior to the assay: withholding IL-2 feeding in Fc-stimulated PBMCprevented the expansion of non-Fc-specific Treg and/or the expansion ofeffector T cell stimulated via Fcγ receptors. On day 4, culturesupernatants were collected to measure IL-10, IFNγ and IL-2 by ELISA andto perform FACS analysis as described below.

CD4+ CD25^(high) T cell surface phenotype was determined by stainingwith specific monoclonal antibodies: anti-CD4 PerCP-Cy5.5, mouse IgG1κ,clone RPA-T4 and anti-CD25 PE, mouse IgG1κ, clone BC96 from eBioscience.BD FACSCanto was used for data acquisition; data were analyzed withFACSDiva (BD Biosciences) or FlowJo software (Tree Star, Inc).Intracellular FOXP3+ was measured with a kit from eBioscience(anti-human FOXP3 PE, mouse IgG1κ, clone 259/D/C7, anti-CD4 FITC, mouseIgG1κ, clone RPA-T4 and anti-CD25 APC, mouse IgG1κ, clone M-A251).Surface anti-IL-7r was measured using anti-CD127 PE, mouse IgG1κ, cloneHIL-7R-M21; CD45RA by using anti-CD45RA-APC, mouse IgG2bκ, clone HI100from BD Bioscience; IL-15r with anti-IL15r FITC, mouse IgG2b, cloneJM74A; CCR7 with anti-CCR7 PE, rat IgG2aκ, clone 3D12; CCR6 withanti-CCR6 PE-Cy7, mouse IgG1κ, clone R6H1; CCR4 with anti-CCR4 PE, mouseIgG1κ, clone 1G1 from eBioscience. Levels of IL-10 and IL-4 produced byPBMC, Treg lines, and Treg clones were measured by ELISA with primaryand secondary antibodies from BD Bioscience. mRNA was extracted fromTreg clones using TRIZOL according to manufacturer's instructions. mRNAtranscript abundance levels from cDNA derived from 25 ng of total RNAwere measured using TAQMAN™ 5′-nuclease gene expression assay (AppliedBiosystem, Foster City, Calif.) for IL-10, IL-4, TGFβ, IL-17 and CTLA-4.Results were normalized for the housekeeping gene TAF1b.

Activated and Memory CD4+ and CD8+ T Cell Characterization

Activated T cells were detected by staining with anti-human HLA-DR, PE,clone LN3, mouse IgG2b,κ, in combination with anti-human CD4,PerCP-Cy5.5, clone RPA-T4, Mouse IgG1,κ, and anti-human CD8, APC, cloneRPA-T8, Mouse IgG1,κ from eBioscience. Memory T cells were detected withanti-human IL-15Rα, FITC, clone eBioJM7A4, Mouse IgG2b, from BDBioscience.

Treg Cloning and Expansion

Fc-specific Treg lines were generated from PBMC stimulated with purifiedFc (100 μg/ml). T cell lines were established by plating 4×10⁵cells/well in 96 flat-bottomed plates (Falcon). Each well was evaluatedas an individual T cell line. On day 4 after stimulation in vitro, cellcultures were fed 100 U/ml of recombinant IL-2 (Peprotech), expanded fortwo days, and tested for specificity at day 6 from the first Fcstimulation in vitro (25). The specificity of the T cell lines wasdetermined by measuring IL-10 in T cell culture supernatants collectedat 48 hours following stimulation with one of the following: 1)irradiated autologous PBMC (negative control); 2) irradiated autologousPBMC pulsed with 20 μg/ml Fc; 3) irradiated allogeneic PBMC pulsed with20 μg/ml Fc (MHC-restriction control). Treg lines that responded byproducing IL-10 to autologous PBMC pulsed with Fc, but not to autologousPBMC alone or allogeneic PBMC pulsed with Fc, were cloned by limitingdilution (0.3-1c/w) in the presence of 2×10⁴ irradiated autologousEBV-transformed B cells per well pulsed with 20 μg/ml Fc as APC/antigensource. EBV-transformed B cell lines were obtained by infecting PBMCwith supernatant from an EBV-producing marmoset-derived cell line B95-8purchased from American Tissue Culture Collection (ATCC). T cell cloneswere stimulated weekly with irradiated autologous EBV lines pre-pulsedwith Fc fragments and expanded with 100 U/ml of IL-2 every other day.Cloning to obtain IgG− and IgG+ autologous and allogenic B cell lines asAPC source

IgG+ autologous B cells to address the recognition of endogenous Fc byTreg (in addition to exogenous Fc) were obtained from EBV-transformed Bcells derived from acute KD patients cloned early after transformationby limiting dilution. B cell clones were screened for surface IgGexpression using anti-human CD19, APC-cy7, clone SJ25c1, mouse IgG1κ,and anti-human IgG, PE, clone G18-145, Mouse IgG1κ, that recognizes theframework of all IgG isotypes from BD Bioscience.

Statistical Analysis

Data analysis was conducted using PRISM™ version 5.0 software (GRAPHPAD™Software, San Diego, Calif.). Statistical significance of the observedFc-specific Treg expansion versus no antigen control was assessed byWilcoxon matched-pairs signed rank test. Non-parametric Mann-Whitneytest was used to compare the percent increase in Treg between groups ofKD children. p-values less than 0.05 were considered significant.

Results

IL-10 Increases in Culture Supernatants of PBMC from Sub-Acute KDPatients Stimulated with Fc Fragments

To address a possible role of the Fc in stimulating Treg in anantigen-specific, MHC-restricted manner, we developed an assay, startingwith a small number of specific precursors, to measure IL-10 secretionand CD4+ CD25^(high) T cell expansion in response to scalar doses ofpurified Fc fragments (1, 10, 100 μg/ml) in bulk PBMC cultures.

After Ficoll-hypaque separation, PBMC from 15 sub-acute KD subjects whohad received IVIG 2 weeks earlier (Table 1), were plated with scalardoses of purified Fc fragments for 4 days. Culture supernatants werecollected to measure IL-10, IFNγ and IL-2. The measurement of IFNγ andIL-2 was included in these experiments to address a possible expansionof pro-inflammatory effector T cells by Fc stimulation.

IL-10 secretion by PBMC was documented in the culture supernatants ofall 15 sub-acute KD subjects studied after IVIG therapy (subjects 1-14and 20) (FIG. 1), regardless of their coronary artery status. Thehighest concentration of Fc tested (100 μg/ml) was the most effective(FIG. 1 of Example 2). The Fc fragments did not stimulatepro-inflammatory T cells with these experimental condition of Example2).

Fc-Specific Treg Expand Only in KD Patients with Normal CoronaryArteries after IVIG but not in Patients with Arterial AbnormalitiesDespite the Presence of IL-10

To address the importance of the Treg specificity for the Fc ininfluencing clinical outcome, we measured the Treg response to purifiedFc fragments in the same cultures in which IL-10 was detected (FIG. 1 ofExample 2). Under these experimental conditions, only 4 days in culturein the absence of IL-2 in vitro, only Treg responding to the Fc via TcRrecognition can survive and proliferate. 10 KD patients who did notdevelop arterial abnormalities after IVIG therapy (subjects 1-10, FIG.2) showed a brisk expansion of CD4+CD25^(high) T cells after 4 days inresponse to scalar doses of Fc. Notably, Treg from the five patients whodeveloped coronary artery abnormalities failed to respond to Fcstimulation in vitro (subjects 11-14 and 20, FIG. 2 of Example 2).Presumably, the source of the IL-10 in the subjects that developedarterial abnormalities was tolerogenic DC that we found abundant duringthe acute phase of KD and increase in numbers after IVIG in thesub-acute phase regardless from the clinical outcome (4).

When we compared the magnitude of the Fc-specific Treg response in PBMCfrom the acute and sub-acute KD subjects, marked Fc-specific Tregexpansion after IVIG treatment was observed only in patients with normalarteries but not in patients who developed arterial abnormalities (FIG.3 of Example 2).

As shown in FIG. 4, FOXP3 analysis by intracellular staining confirmedthe lack of Treg expansion in Fc-stimulated cultures from a sub-acute KDpatient who developed coronary arterial dilation, subject 15, incontrast to a sub-acute KD patient with normal coronary arteries afterIVIG therapy, subject 16, Table 1. These results further support theconcept that the expansion of Fc-specific Treg plays an important rolein resolution of the acute vasculitis in KD patients.

In five Treg responders, 10 μg/ml Fc was more immunogenic than 100 μg/ml(subjects 1, 2, 3, 4, 9; FIG. 2 of Example 2), in sharp contrast to thehigher dose required to stimulate IL-10 secretion in the same patients(FIG. 1 of Example 2). These results may reflect different MHChaplotypes that bind relevant Fc peptides recognized by Treg withdifferent TcR affinities. The FACS images summarized in FIG. 2 areprovided in supplemental FIG. 1A. As a control for antigenicspecificity, we tested Treg expansion in response to increasingconcentrations of F(ab)₂ fragments and documented no expansion(supplemental FIG. 1B).

Although we were unable to perform classic suppression assays due to thelimited number of cells and the very young age of the patients that didnot complete yet vaccination protocols, we observed that T cells with apro-inflammatory phenotype decreased as the Fc-specific Treg increased.Specifically, activated CD4+ and CD8+DR+ T cells, but not IL-15(receptor)r+ T cells, decreased in Fc-stimulated PBMC cultures in whichthe Treg expanded (subjects 18 and 19, FIG. 5).

Characterization of Fc-Specific Treg Clones Derived after IVIG fromSub-Acute KD Subjects with Normal Coronary Arteries

Treg clones were generated from Fc-specific Treg lines derived fromIVIG-treated, sub-acute KD subjects with normal coronary artery internaldiameters and were screened for specificity. B-cell lines derived from 4KD subjects were cloned by limiting dilution and screened for IgGexpression. We measured IL-10 production by Treg clones in response toirradiated, autologous, IgG-negative B cell lines alone (control) or thesame B cell lines incubated with Fc fragments. We expanded and furthercharacterized the phenotype and lymphokine profile of Treg clones thatmade IL-10 in response to Fc-pulsed autologous IgG-negative B cells butnot to the same B cells without Fc. Treg clones that secreted IL-10 inresponse to autologous B cells in the absence of Fc stimulation wereconsidered to be auto-reactive for self-antigens other than Fc and werenot studied further.

Fc-specific Treg clones produced high amounts of IL-10 and lower amountsof IL-4 but not TGFβ (FIG. 6A). The production of IL-10 and IL-4, butnot TGFβ were confirmed by qRT-PCR analysis of RNA extracted fromindividual clones, which also demonstrated high transcript abundance forCTLA-4, a canonical marker for all Treg lineages, and no expression ofIL-17 (FIG. 6A). T cells were further defined by surface andintracellular markers: CD4+ CD25^(high), IL-7r (CD127)^(low),CD45RA^(low) FOXP3^(high) (FIG. 6B). The memory marker IL-15r and CCR6,the homing receptor for vessels, were not expressed by Fc-specific Tregclones, which did express high levels of CCR7, an important homingreceptor for lymphoid organs (FIG. 6B). The low expression level ofCD127 coupled with absent expression of CCR4 excluded the possibility ofcontamination by T helper (h) 2 cells in this T cell clonal effort (26).

Fc-Specific Treg Clones Respond to Autologous IgG+ B Cells in theAbsence of Exogenous Fc

We next addressed whether Fc-specific Treg clones respond to both: 1)exogenous Fc fragments presented by autologous IgG negative B cells and2) endogenous Fc derived from membrane IgG on the cell surface ofautologous IgG+ B cells. When autologous, live, irradiated IgG+ B cellswere used as antigen presenting cells, Fc-specific Treg clones respondedby secreting IL-10 and IL-4 in the absence of exogenous Fc (FIG. 7A). Todetermine if conventional antigen processing of IgG molecules by B cellsis required to stimulate Fc-specific Treg, we tested in parallel theantigen presenting capacity of live versus paraformaldehyde-fixedautologous IgG+ B cell lines (FIG. 7A). MHC restriction was required forFc presentation and Treg response because only autologous, but notallogeneic, IgG+ B-cells induced IL-10 and IL-4 secretion (FIG. 7B). Therequirement for TcR signaling rather than Fcγ receptor stimulation on Tcells was further confirmed by the dose-dependent IL-10 production byTreg clones in response to an anti-CD3 agonistic antibody (supplementalFIG. 2).

Treg Expand when Stimulated In Vitro by Fc in Children with AcuteInfections and Drug Reactions, but not in Untreated Children withAutoimmune Diseases

To address if this Fc-specific Treg population physiologically expandduring other acute pediatric inflammatory conditions, we tested themagnitude of Treg expansion in Fc-stimulated PBMC in a variety ofcontrols that included children with self-limited viral infections(n=7), bacterial infections (n=2), systemic drug reaction (n=1), andthree newly diagnosed, untreated autoimmune diseases (juvenileidiopathic arthritis, n=2, Henoch Schoenlein purpura, n=1). In 10 of 13pediatric subjects with acute viral or bacterial infection or systemicdrug reactions, but not autoimmunity, Fc-induced Treg responses weredetected (median Treg %=0.28%, 95% CI 0.20-0.37 at 10 μg/ml of purifiedFc) (FIG. 8).

Discussion

There is a growing list of the immune modulatory functions of IVIG thatincludes expansion of IL-10-secreting DC stimulated by Fc fragments andthe non-specific, IL-10 mediated expansion of Treg observed in humansand mice (3, 4, 6, 27, 28). To this list, we can now add a newlydiscovered action of IVIG: expansion of a population of Treg thatrecognize the Fc region of IgG presented in a conventionalMHC-restricted, TcR-mediated fashion by mature B cells. Following IVIGtherapy, this Fc-specific Treg population expands in KD patients withnormal coronary arteries but fails to expand in patients who developdilated or aneurysmal coronary arteries despite IVIG treatment. AlthoughIL-10 secretion in response to Fc stimulation of PBMC in sub-acute KDpatients after IVIG is important, it is not sufficient to resolve thevasculitis, since in the absence of Fc-specific Treg expansion followingin vivo boost by IVIG therapy it did not prevent coronary arterialabnormalities. Our results demonstrate that the specificity of the Treg,as for any other adaptive immune response, is critical for thesuppression in vivo of pathogenic T cells. This Treg population expandsduring acute inflammation in patients suffering from a variety ofdifferent infections, but not in children with new-onset, untreatedautoimmune disease.

The recognition of endogenous IgG presented by mature B cells appears tomaintain Fc-specific Treg that have been activated by IVIG in vivo.These Treg are fully functional in very young infants and children. Bcell antigen presentation of endogenous Fc by mature B cells andcirculating IgG presented by conventional APC (i.e. DC) keep stimulatingFc-specific Treg: the antigen (Fc) is always present under physiologicalconditions and IgG-expressing mature B cells reside in normal lymphnodes, tonsils, Peyer patches, bone marrow and other secondary lymphoidorgans.

The phenotype of Fc-specific Treg clones that expand after IVIG in KDpatients suggests that they down-regulate acute vasculitis bysuppressing pro-inflammatory T cells not in the inflamed coronaryarteries but in the lymph nodes and other secondary lymphoid organs asevidenced by the expression of CCR7 (19). In fact, CCR6, which is acritical chemokine receptor for T cell homing to the vessels (29), wasnot expressed on any of the Treg clones studied. Lack of CCR6 expressionon Fc-specific Treg clones further supports the idea that this T cellsubset is different from auto-reactive, peripherally induced Tregpreviously reported in humans (19) or pathogen-induced human Th17 Tcells that produce IL-10 regulated by IL-1 (30). The Treg populationdescribed herein is also different from the Treg that respond tooptimized, pan-DR-binding exogenous Fc peptides (Tregitopes), which havebeen previously described in adult healthy donors (24). Those T cellswere CCR7 negative and did not express high levels of CTLA-4, which wedetected in all the Fc-specific Treg clones studied. Moreover, the Tregdescribed here secrete low amounts of IL-4 that may be functionallyimportant to sustain B cell survival and expansion. The results suggestin fact a new model of T-B cooperation.

An important role for Fc-specific Treg and mature B cell-Treginteractions in immune-regulation is further supported by the bluntedexpansion of this Treg specificity in children with newly diagnosed,untreated autoimmune diseases. Collectively, it appears as if a defectin the Treg-mediated suppression of the pro-inflammatory T cell responsein KD patients with coronary artery abnormalities and in children withautoimmune disorders is associated with disease progression.

In summary, we demonstrate the role of exogenous IgG and mature B cellsin the control of an acute pediatric vasculitis of the coronary arteriesvia the expansion of Treg that recognize the Fc portion of IgG. Ourresults suggest a novel mechanism for the anti-inflammatory action andclinical benefit provided by IVIG therapy in patients with KD and,potentially, other vasculitides (34). Measuring the expansion ofFc-specific Treg may be helpful for clinical risk assessment in the caseof KD and potentially for monitoring other immune-mediated pathologicalconditions and response to IVIG therapy.

Figure Legends—Example 2 FIG. 1 of Example 2 (or FIG. 2).

PBMC derived from sub-acute KD subjects secrete IL-10, but not IFN-gammaor IL-2 in response to scalar doses of Fc. PBMC from 15 KD subjects, 10with normal arteries after IVIG therapy (subjects 1-10) and 5 withdilated arteries or aneurysm after IVIG therapy

(subjects 11-14 and 20) were cultured with scalar doses of purified Fcfragments (Meridian Life Science, purity>97%) for 4 days in the absenceof exogenous lymphokines. Supernatants were harvested and tested forIL-10, IFN-gamma and IL-2 secretion by ELISA.

FIG. 2 of Example 2 (or FIG. 3).

Enumeration of Fc-induced Treg in sub-acute KD patients with normalarteries after IVIG. FIG. 2A: CD4+ CD25^(high) Treg expansion to scalardoses of Fc was analyzed by flow cytometry in PBMC cultures derived from15 sub-acute KD subjects, with normal arteries (subjects 1-10) and 5with dilated arteries or aneurysm (subjects 11-14 and 20) previouslystudied for lymphokine production. PBMC were cultured for 4 days with 0(no antigen), 1, 10, or 100<g/ml of purified Fc fragments (Meridian LifeScience, purity≥97%). FIG. 2B: Summary of the Treg response in 15sub-acute KD subjects described in panel A. The median % increase inTreg expansion for the subjects with normal coronary arteries wascalculated with an Fc dose of 10 μg/ml.

FIG. 3 of Example 2 (or FIG. 4).

Differential expansion of Fc-specific Treg from the acute to thesub-acute phase of KD depending upon clinical outcome. We studied thefold-increase of the Fc-specific Treg repertoire from the acute tosub-acute phase in 6 KD subjects: three subjects with normal coronaryarteries after IVIG therapy (subjects 2, 7, 10), three subjects withdilated arteries or aneurysm despite IVIG therapy (subjects 12, 13, 14).

FIG. 4 of Example 2 (or FIG. 5).

FOXP3+ T cells expansion in response to Fc. Intracellular staining forFOXP3^(high) expression in CD4+ T cells was assessed in Fc-stimulatedPBMC cultures from two subacute patients after IVIG therapy. Subject 15had dilated coronary arteries by echocardiogram, and there was noexpansion of the Fc-specific Treg population. Subject 16 had normalcoronary arteries and showed expansion of the Treg population inresponse to 100<g/ml Fc.

FIG. 5 of Example 2 (or FIG. 6).

Activated CD4+ and CD8+ CD25^(low) DR^(high) but not memory IL15r+ Tcells decrease in PBMC cultured with Fc when Treg expand. PBMC from twosub-acute KD subjects with normal arteries after IVIG (Subjects 18 and19) were cultured with 100<g/ml purified Fc fragments or media alone ascontrol to address the fate of activated T cells and memory T cells inFc-stimulated cultures in which Treg expand. Treg were defined as CD4+CD25^(high); activated CD4+ and CD8+ T cells as CD25^(low) andDR^(high); and memory T cells as IL-15r+. After 4 days in culture withFc, Treg expanded but activated CD4+ and CD8+ T cell numbers decreased.IL-15r+ memory T cells remained unchanged.

FIG. 6 of Example 2 (or FIG. 7).

Characterization of Fc-specific Treg clones. Three representative Tregclones generated from KD subject 17 two weeks following IVIG treatment.Panel A: Left: Production of IL-10, IL-4 and TGFβ measured by ELISA 48hours after stimulation with autologous, irradiated B cells pulsed with20 μg/ml Fc fragments; Right: qRT-PCR analysis of cell lysates from thesame three Treg clones. Panel B: Phenotype of a representative Tregclone characterized as CD4+ CD25^(high) intracellular FOXP3^(high),CD45RA^(low), IL-7r^(low), IL15r−, CCR6−, CCR7^(high) and CCR4−.

FIG. 7 of Example 2 (or FIG. 8).

Fc-specific Treg clones recognize endogenous processed IgG presented byautologous B cells in an MHC-restricted, TcR-mediated manner. Panel A:Live, but not paraformaldehyde-fixed, autologous EBV-transformed IgG+ Bcells stimulate Treg clones to secrete IL-10 and IL-4 in the absence ofexogenous Fc demonstrating that a) IgG needs to be conventionallyprocessed for Treg recognition; b) the Treg response is not directed toself MHC molecules. Panel B: Only autologous, but not allogeneic IgG+ Bcells activate a Fc-specific Treg clone, demonstrating that theFc-specific Treg response is MHC-restricted.

FIG. 8 of Example 2 (or FIG. 9).

Treg expand in response to Fc in acute pediatric inflammatory conditionsbut not in subjects with acute autoimmune conditions. Fc-induced Tregexpansion in cultured PBMC was detected after Fc stimulation in 7subjects with acute viral infections, 2 subjects with bacterialinfections and 1 subject with a systemic drug reaction. Three patientswith new-onset, untreated autoimmune conditions failed to show Tregexpansion in response to Fc.

Supplemental FIG. 1 of Example 2 (or FIG. 10).

Treg response to purified Fc and F(ab)2 fragments in sub-acute KDsubjects. Panel A: CD4+ CD25^(high) Treg expansion in PBMC cultures fromsub-acute KD subjects (subjects 1-10) with normal arteries after IVIGtherapy in response to scalar doses of Fc. Panel B: Lack of CD4+CD25^(high) Treg expansion in response to F(ab)2 fragments in 4 patientswithin the same cohort (subjects 1, 2, 4, 7).

Supplemental FIG. 2 of Example 2 (or FIG. 11).

The Fc-specific clonal Treg response is TcR mediated. Two representativeFc-specific Treg clones secrete IL-10 when stimulated 24 hours in vitrowith an anti-CD3 agonistic antibody. Fc stimulates Treg production ofIL-10 when presented by autologous APC. Fc alone does not stimulateIL-10 production.

Example 3: Peptides Effective for Expanding Regulatory T Cells (Treg)Populations—Fine Specificities of Natural Regulatory T Cells after IVIGTherapy in Patients with Kawasaki Disease—Epitope Mapping of HumanFc-Specific Natural Treg

This example presents data demonstrating that compositions as providedherein effectively expand regulatory T cells (Treg) populations for,e.g., ameliorating vascular inflammation, and Kawasaki disease or apediatric acute vasculitis of the coronary arteries, including vascularcoronary abnormalities and the same or similar types of acute or chronicvascular inflammatory abnormalities.

Here we characterize the fine specificity of nTreg in sub-acute KDsubjects (2-8 weeks post-IVIG) and convalescent KD subjects (1-10 yearspost-IVIG) by testing the immunogenicity of 64 peptides, 15 amino acidsin length with a 10 amino acid-overlap spanning the entire Fc protein.Overall, 12 Fc peptides (6 pools of 2 consecutive peptides) wererecognized by nTreg in the cohorts studied, including two patients withCAA.

To test whether IVIG stimulates the expansion of the same nTregpopulations that maintain vascular homeostasis under physiologicalconditions in healthy subjects, we compared these results with resultsobtained in healthy adult controls. Similar nTreg fine specificitieswere observed in KD patients after IVIG and in healthy donors. Overall,these results suggest that T cell fitness rather than T cell clonaldeletion or anergy is responsible for the lack of Fc-specific nTregexpansion in KD patients who develop CAA.

Furthermore, we found that adolescents and adults who had KD duringchildhood without developing CAA did not respond to the Fc protein invitro, suggesting that the nTreg response induced by IVIG in KD patientsis short-lived. Our results support the concept that synthetic,Fc-derived peptide epitopes as provided herein ar a viable therapeuticapproach to expand Fc-specific nTreg and more effectively prevent CAA inKD patients.

In this study, we describe the fine specificity of Fc-specific nTreg bytesting their response to overlapping peptides covering the entire Fcmolecule. We also tested the nTreg response to the whole Fc protein ofadolescents and adults with a history of KD in childhood to assess thedurability of the nTreg response years after IVIG and we compared itwith sex-matched healthy donors. These studies showed that Fc-specificnTreg fine specificity is similar in KD and healthy donors, but theseresponses are short lived in KD patients. Since this defect can beovercome by administration of large doses of IVIG in most KD patients,our results demonstrate that the administration of Fc-derived peptideepitopes as provided herein are a viable therapeutic approach to expandFc-specific nTreg and prevent CAA.

Material and Methods Study Population:

Sub-acute and convalescent pediatric KD patients were enrolled at RadyChildren's Hospital San Diego following parental informed consent andpatient assent as appropriate. All KD subjects were treated with IVIG 2g/kg and aspirin 80-100 mg/kg/day until afebrile, then 3-5 mg/kg/dayuntil the platelet count had returned to normal. All sub-acute subjectswere taking low dose aspirin at the time of phlebotomy. KD subjects (10sub-acute subjects: 5 males, 5 females aged 2.0-15.5 years at time ofstudy) and six convalescent subjects: 5 males, 1 female, aged 2.4-15.7years at time of study) were evaluated by echocardiography during theacute admission and at 2 and 6 weeks and 1 year following diagnosis. Theinternal diameter of the right and left anterior descending coronaryarteries was measured and expressed as a Z score (standard deviationunits from the mean normalized for body surface area; normal Zscore<2.5). Z_(worst) was defined as the highest Z score of eithercoronary artery measured during the first 6 weeks after fever onset. Twoof the subacute patients developed CAA despite IVIG treatment, asillustrated in Table 1, or FIG. 12, which illustrates the demographicand clinical status of pediatric KD study subjects. Heparinized bloodsamples (1-4 ml) were obtained 10-54 days post-IVIG (sub-acute cohort,subjects #1-10) and 1-2 years post-IVIG for five subjects (#11-14, 16)and 10 years post-IVIG for one subject (#15) (convalescent cohort).

As a comparison group for the Fc epitope mapping, six normal healthydonors (4 males, 2 females aged 25-59 years) were recruited at theScripps Research Institute, La Jolla Calif. following written informedconsent (IRB#101213X).

To test the durability of the Fc-specific nTreg response, 8 subjects whohad KD during childhood without developing CAA (4 males and 4 femalesaged 12-42 years, Table 2, below) were tested for Fc-specific nTregresponses to the whole Fc protein in vitro and compared to 8 additionalsex-matched healthy donors.

TABLE 2 Demographic characteristics of adolescent and adult subjectswith a history of KD in childhood. KD Age at KD Age at testing Race/IVIG subject # Sex onset (years) (years) ethnicity treatment A1 M 7 19White Yes A2 M 3 19 Hispanic Yes A3 F 1 12 African- Yes American A4 M 416 White Yes A5 F 5 28 White Yes A6 M 2 19 Mixed No A7 F 4 21 AsianUnknown A8 F 1 42 Asian No

Peptides:

Peptides were synthesized by Fmoc chemistry using a multiplex peptidesynthesizer (Symphony X, Protein Technologies Inc., Tucson, Ariz.).Peptides were cleaved automatically on the synthesizer usingtrifluoroacetic acid. Peptides were ≥97% pure as assessed by C18 reversephase HPLC, and the identity of the peptides was verified by massspectrometry. A total of 64 peptides, each 15 amino acids in length witha 10 amino acid-overlap for each peptide, spanning the whole Fc moleculewere used to define the fine specificity of Fc-specific nTreg. The aminoacid sequences of the 15-mer overlapping peptides are shown in Table 3,see FIG. 13; and also summarized (** indicates an exemplary peptidesequence of the invention):

Fc position 1-15 (SEQ ID NO: 18) STKGPSVFPLAPSSK Fc position 6-20 (SEQID NO: 19) SVFPLAPSSKSTSGG Fc position 11-25 (SEQ ID NO: 20)APSSKSTSGGTAALG Fc position 16-30 (SEQ ID NO: 21) STSGGTAALGCLVKD ** Fcposition 21-35 (SEQ ID NO: 1) TAALGCLVKDYFPEP ** Fc position 26-40 (SEQID NO: 2) CLVKDYFPEPVTVSW Fc position 31-45 (SEQ ID NO: 22)YFPEPVTVSWNSGAL Fc position 36-50 (SEQ ID NO: 23) VTVSWNSGALTSGVH Fcposition 41-55 (SEQ ID NO: 24) NSGALTSGVHTFPAV Fc position 46-60 (SEQ IDNO: 25) TSGVHTFPAVLQSSG ** Fc position 51-65 (SEQ ID NO: 9)TFPAVLQSSGLYSLS ** Fc position 56-70 (SEQ ID NO: 10) LQSSGLYSLSSVVTV **Fc position 61-75 (SEQ ID NO: 11) LYSLSSVVTVPSSSL ** Fc position 66-80(SEQ ID NO: 12) SVVTVPSSSLGTQTY Fc position 71-85 (SEQ ID NO: 26)PSSSLGTQTYICNVN Fc position 76-90 (SEQ ID NO: 27) GTQTYICNVNHKPSN Fcposition 81-95 (SEQ ID NO: 28) ICNVNHKPSNTKVDK Fc position 86-100 (SEQID NO: 29) HKPSNTKVDKKVEPK Fc position 91-105 (SEQ ID NO: 30)TKVDKKVEPKSCDKT Fc position 96-110 (SEQ ID NO: 31) KVEPKSCDKTHTCPP Fcposition 101-115 (SEQ ID NO: 32) SCDKTHTCPPCPAPE Fc position 106-120(SEQ ID NO: 33) HTCPPCPAPELLGGP Fc position 111-125 (SEQ ID NO: 34)CPAPELLGGPSVFLF Fc position 116-130 (SEQ ID NO: 35) LLGGPSVFLFPPKPK **Fc position 126-140 (SEQ ID NO: 4) PPKPKDTLMISRTPE Fc position 131-145(SEQ ID NO: 36) DTLMISRTPEVTCVV Fc position 136-150 (SEQ ID NO: 37)SRTPEVTCVVVDVSH Fc position 141-155 (SEQ ID NO: 38) VTCVVVDVSHEDPEV Fcposition 146-160 (SEQ ID NO: 39) VDVSHEDPEVKFNWY Fc position 151-165(SEQ ID NO: 40) EDPEVKFNWYVDGVE Fc position 156-170 (SEQ ID NO: 41)KFNWYVDGVEVHNAK Fc position 161-175 (SEQ ID NO: 42) VDGVEVHNAKTKPRE Fcposition 166-180 (SEQ ID NO: 43) VHNAKTKPREEQYNS Fc position 171-185(SEQ ID NO: 44) TKPREEQYNSTYRVV ** Fc position 176-190 (SEQ ID NO: 13)EQYNSTYRVVSVLTV ** Fc position 181-195 (SEQ ID NO: 14) TYRVVSVLTVLHQDWFc position 186-200 (SEQ ID NO: 45) SVLTVLHQDWLNGKE Fc position 191-205(SEQ ID NO: 46) LHQDWLNGKEYKCKV Fc position 196-210 (SEQ ID NO: 47)LNGKEYKCKVSNKAL Fc position 206-220 (SEQ ID NO: 48) SNKALPAPIEKTISK Fcposition 211-225 (SEQ ID NO: 49) PAPIEKTISKAKGQP Fc position 216-230(SEQ ID NO: 50) KTISKAKGQPREPQV Fc position 221-235 (SEQ ID NO: 51)AKGQPREPQVYTLPP Fc position 226-240 (SEQ ID NO: 52) REPQVYTLPPSRDEL Fcposition 231-245 (SEQ ID NO: 53) YTLPPSRDELTKNQV Fc position 236-250(SEQ ID NO: 54) SRDELTKNQVSLTCL Fc position 241-255 (SEQ ID NO: 55)TKNQVSLTCLVKGFY Fc position 246-260 (SEQ ID NO: 56) SLTCLVKGFYPSDIA Fcposition 251-265 (SEQ ID NO: 57) VKGFYPSDIAVEWES Fc position 256-270(SEQ ID NO: 58) PSDIAVEWESNGQPE Fc position 261-275 (SEQ ID NO: 59)VEWESNGQPENNYKT ** Fc position 266-280 (SEQ ID NO: 7) NGQPENNYKTTPPVL **Fc position 271-285 (SEQ ID NO: 8) NNYKTTPPVLDSDGS ** Fc position276-290 (SEQ ID NO: 15) TPPVLDSDGSFFLYS Fc position 281-295 (SEQ ID NO:60) DSDGSFFLYSKLTVD Fc position 286-300 (SEQ ID NO: 61) FFLYSKLTVDKSRWQ** Fc position 291-305 (SEQ ID NO: 5) KLTVDKSRWQQGNVF ** Fc position296-310 (SEQ ID NO: 6) KSRWQQGNVFSCSVM ** Fc position 301-315 (SEQ IDNO: 16) QGNVFSCSVMHEALH ** Fc position 306-320 (SEQ ID NO: 17)SCSVMHEALHNHYTQ Fc position 311-325 (SEQ ID NO: 62) HEALHNHYTQKSLSL Fcposition 316-329 (SEQ ID NO: 63) NHYTQKSLSLSPGKCharacterization of Peptide-Specific nTreg Responses:

Heparinized blood samples were collected in sodium heparin green toptubes for isolation of peripheral blood mononuclear cells (PBMC) fromhealthy adult donors and KD subjects. To enumerate Fc-specific nTregthat expand after IVIG infusion, we previously developed a method toavoid non-specific expansion of peripherally-induced (p)Treg bytolerogenic dendritic cells (DC) or the expansion of effector T cells(3). PBMC were plated with scalar doses of purified Fc (1, 10 and 100μg/ml; Life Meridian Science) or pools containing two peptides each (20μg/ml/peptide, Table 3, see FIG. 13) at a concentration of 2×10⁵cells/well in 96-well flat-bottomed plates (Falcon) for 4 days. Cellcultures did not receive any exogenous IL-2 prior to the assays toprevent the expansion of non-Fc-specific nTreg and/or the expansion ofeffector T cells stimulated via Fc-gamma receptors.

On culture day 4, supernatants were collected to measure IL-10 secretionby ELISA with primary and secondary antibodies from BD Bioscience on allsubjects as previously described (3). A positive nTreg response wasdefined as IL-10 secretion that exceeded 20 pg/ml. For four KD subjectsand all eight of the normal adult donors, cells from these cultures wereharvested on day 4 for FACS analysis. CD4+ CD25^(high)T cell surfacephenotype was determined by staining with specific monoclonalantibodies: anti-CD4 PerCP-Cy5.5, mouse IgG1 κ, clone RPA-T4 andanti-CD25 PE, mouse IgG1κ, clone BC96 from eBioscience. BD FACSCanto wasused for data acquisition; data were analyzed with FACSDiva (BDBiosciences) or FlowJo software (Tree Star, Inc).

HLA Class II Binding Prediction for Fc-Derived Peptides

As a preliminary analysis to determine the potential HLA class IIbinding capacity of Fc-derived peptides, we scanned the sequences usingthe suite of class II algorithms available through the NIH-funded and LaJolla Institute for Allergy and Immunology-administered website, ImmuneEpitope Database (IEDB). For HLA class II binding predictions, theentire IgG Fc sequence was parsed into the set of 15-mer peptides,overlapping by 10 residues (Table 3, FIG. 13, and see summary, above)that we functionally tested for immunogenicity in KD subjects after IVIGand healthy donors. The capacity of each peptide to bind a panel of 27common HLA class II DR, DQ and DP molecules was predicted using the IEDBconsensus algorithm, see e.g., Vita et al., The immune epitope database(IEDB) 3.0. Nucleic Acids Res. 2014 Oct. 9. (www.iedb.org<http://www.iedb.org>) (8). A peptide was considered a binder to anyclass II if its corresponding consensus score was ≤20^(th) percentile(8). Binding to any specific allele was defined as a correspondingconsensus prediction score≤20th percentile. The number of alleles boundwas tabulated for each peptide. A promiscuous binding peptide wasoperationally defined as a peptide binding or predicted to bind to 50%or more of the 26 alleles. The total number of class II moleculespredicted to be bound was tabulated and plotted.

Results

Fine Specificity of Fc-Specific nTreg Determined with 15-Mer OverlappingPeptides in KD Subjects after IVIG

Discrete regions within the Fc sequence were immunogenic for nTreg insubacute KD subjects after IVIG stimulating IL-10 secretion in 9 of the10 subjects and including CD4+ CD25^(high) T cell expansion in all foursubjects assessed (subject 1 recognized a unique set of epitopes, datanot shown). Peptides were ranked based on IL-10 secretion and peptides121-135 and 126-140 (pool 13) were the most immunogenic being recognizedby nTreg in 8 of 16 (50%) KD subjects (FIG. 1 of Example 3, or FIG. 14).Other sequences also resulted in expansion of the nTreg populations: 7of 16 (44%) KD subjects responded to amino acid residues 276-290 and281-295 (pool 28); three different peptide pools corresponding aminoacid residues 51-65 and 56-70 (pool 6), amino acid residues 181-195 and186-200 (pool 19), amino acid residues 301-315 and 306-320 (pool 31)were recognized in 6 of 16 (37%) KD subjects. Finally, 5 out of 16 (31%)KD subjects recognized amino acid residues 61-75 and 66-80 (pool 7). Ofinterest, the two KD subjects with CAA (subjects #5 and 6) showed ameasurable nTreg response to pool 28 and pool 6, respectively (FIG. 1 ofExample 3, or FIG. 14), suggesting that the fine specificity of this KDsubgroup was similar to the remainder of the KD subjects tested.

The strength of the response varied among subjects possibly reflectingtheir HLA type and the precursor frequency of peptide-specific nTregafter IVIG. We observed a rapid expansion of the CD4+ CD25^(high) T cellpopulation in some KD patients in response to peptide epitopes afterIVIG, suggesting a very high precursor frequency (FIG. 2 of Example 3,or FIG. 15).

Fine Specificity of Fc-Specific nTreg Determined with 15-Mer OverlappingPeptides in Healthy Adult Donors

To compare Fc-specific nTreg responses that arise in KD patients afterIVIG to the specificities of nTreg in healthy donors, we performed Fcepitope mapping in six healthy adult subjects. As observed in KDpatients after IVIG, discrete regions within the Fc sequence were foundto be immunogenic for nTreg and stimulated the secretion of IL-10 andCD4+ CD25^(high) T cell expansion. The epitope ranking indicated thatthe immunodominant responses were similar in healthy controls comparedto KD patients after IVIG. Peptides 121-135 and 126-140 (pool 13) werethe most immunogenic and were recognized by nTreg in 6 of 7 (86%) donors(FIG. 3 of Example 3, or FIG. 16). Several other pools defined asimmunogenic in KD subjects also stimulated nTreg in healthy donors: 5 of7 (71%) healthy donors responded to amino acid residues 276-290 and281-295 (pool 28), 301-315 and 306-320 (pool 31); 4 of 7 (57%) donorsresponded to amino acid residues 181-195 and 186-200 (pool 19); 3 of 7(43%) donors responded to amino acid residues 51-65 and 56-70 (pool 6),61-75 and 66-80 (pool 7) ((FIG. 3 of Example 3, or FIG. 16). Responsesto amino acid residues 21-35 and 26-40 (pool 3) and 31-45 and 36-50(pool 4) were unique, as these pools were not immunogenic for the KDsubjects (FIG. 4 of Example 3, or FIG. 17).

nTreg from Healthy Adults Who had KD in Childhood without Developing CAAFail to Respond In Vitro to Whole Fc Protein

We previously showed that acute KD subjects lack Fc-specific nTreg priorto IVIG (3). IVIG therapy activates this nTreg repertoire in KD patientswho do not develop CAA but it was unknown if the Fc-specific nTregresponse to the whole Fc protein was long lasting or if it waned overtime. Therefore, we tested CD4+ CD25^(high) nTreg expansion in responseto Fc fragments in eight healthy adults who had KD during childhoodwithout developing arterial complications (Table 2, see above) andcompared their response with eight healthy adult donors. nTreg derivedfrom adolescents and adults years after IVIG treatment for KD did notexpand in response to Fc fragments in sharp contrast to healthy adultcontrols (FIG. 5 of Example 3, or FIG. 18).

HLA/Fc-Derived Peptide Binding Prediction Analysis

It was previously shown that peptides predicted to bind 50% or more ofthe HLA alleles assessed at the 20% or better consensus prediction levelcorrespond to the most dominant epitopes, and encompass a large fractionof the T cell responses to EPO (9), allergens such as timothy grass andmycobacteria tuberculosis antigens (10-12). Since peptide:HLA binding isrequired, but not sufficient, to elicit an HLA class II restrictedresponse, peptide:HLA binding predictions do not necessarily predictwhich peptides will be immunogenic as dominant epitopes tend to bepromiscuous binders (10, 13, 14).

Accordingly, the capacity of Fc peptides to bind a panel of 26 HLA classII alleles that are commonly expressed in representative ethnicitiesworldwide and in the US and for which epitope prediction tools are wellestablished and publically available (8, 15), were predicted using toolsavailable through IEDB (www.iedb.org). Three major promiscuous bindingregions were identified, corresponding to residues 181-185 51-56 and56-70 (pool 6), 181-195 and 186-200 (pool 19), 271-285 and 276-290 (pool28) (FIG. 6 of Example 3, or FIG. 19).

These epitopes were compared with the HLA binding peptide patternsidentified by the predicted binding analysis. Of the top 4 immunogenicpools, 3 corresponded to promiscuous binders (i.e. with predictedbinding to ≥13 HLA alleles). Interestingly, pool 13 (peptides startingin positions 121 and 126), which was the most immunogenic, was notassociated with predicted promiscuous binding. In fact, peptide 121 waspredicted to bind only 3 alleles (HLA DRB1*0301, DRB1*1101 andDRB5*0101), and peptide 126 was not predicted to bind any. It ispossible that this region might be associated with restriction byspecific HLA alleles (monogamous restriction) that are present with highfrequency in the donor cohort, and/or that were not present in theallele panel utilized for predictions.

Discussion

In the present study we mapped the Fc peptide regions recognized bynTreg from sub-acute and convalescent KD subjects after IVIG therapy,from adults with a history of KD in childhood, and from healthy adultsubjects. Using IL-10 secretion and CD4+ CD25^(high) T cellproliferation as a read-out, we defined discrete Fc regions that areimmunogenic. Our previous work suggested that immune-regulation viaFc-specific nTreg influences the clinical fate of KD patients andidentified this as one mechanism by which IVIG leads to clinicalimprovement in these patients (3). However, 20-30% of KD patients go onto develop CAA with transient dilation or aneurysms despite timelytreatment with IVIG and their nTreg do not respond to Fc stimulation invitro (3, 4, 16).

By using Fc peptide epitopes as immunogens rather than the whole Fcprotein, we demonstrated that nTreg from patients with CAA can expand invitro (FIG. 1). These results suggest that T cell fitness, rather than Tcell anergy or T cell clonal deletion causes the lack of Fc-specificresponse in KD patients with CAA who do not appear to respond to thewhole Fc protein (3). Thus, the immunodominant Fc sequences recognizedin the cohorts, i.e., the exemplary peptides as provided herein, can beused therapeutic agents. These exemplary peptides as provided herein canhave significant advantages over whole IVIG in terms of safety, productreproducibility, and ease of definition and characterization. Thepredicted HLA binding analysis reported here supports the idea that mostof the immunodominant Fc sequences, including exemplary peptides asprovided herein, and recognized in the cohorts studied, will bindmultiple HLA alleles and can be used as therapeutic peptides.

Among the Fc peptides identified as relevant for nTreg expansion afterIVIG in KD patients, some include portions of the two long Fc sequencespreviously described as Tregitopes, pan-DR epitopes that wereimmunogenic in normal donors (17). Our results confirm the relevance ofthese Fc peptides in expanding regulatory T cells and their pan-DRpotential binding capacity, but also suggest that the Tregitopes are notthe most immunogenic sequences of the Fc protein for nTreg generation(FIGS. 1 and 3 of Example 3). Moreover, our data suggest that theTregitopes are further trimmed for optimal HLA binding and TcRrecognition since positions 51-65 and 56-60 (pool 6) contained withinTregitope 167 and positions 181-195 and 186-200 (pool 19) containedwithin Tregitope 289 were more immunogenic than other amino acidsequences within the two Tregitopes.

These studies suggest that Fc-specific nTreg responses in KD can beelicited by administration of large doses of IVIG in most patients.However, this regulatory T cell response is not long lasting and wasundetectable in adult years after IVIG administration (FIG. 5). Wetherefore postulate a focal immune regulatory defect in patients whodevelop KD that can be overcome in the majority of patients byadministration of a large dose of IVIG.

Figure Legends Example 3 FIG. 1 of Example 3 (or FIG. 14):

nTreg fine specificities in sub-acute and convalescent KD subjects.2×10⁵ PBMC/well derived from 10 sub-acute and 6 convalescent KD subjectswere cultured with pools of two Fc peptides (Table 3, see FIG. 13) for 4days in the absence of exogenous lymphokines. IL-10 secretion inresponse to peptide stimulation was measured in culture supernatants byELISA on day 4. Subjects 5 and 6 both developed CAA and IL-10 secretionwas noted in their PBMC cultures incubated with peptides from Pools 28and 6, respectively.

FIG. 2 of Example 3 (or FIG. 15):

Response of nTreg to peptide pool 13. Enumeration of CD4+ CD25^(high) Tcells from KD subject #10 in response to Fc 121-135 and 126-140 (pool13), the most immunogenic sequences in this cohort of patients (P7,upper right).

FIG. 3 of Example 3 (or FIG. 16): IL-10 secretion in PBMC cultures fromhealthy adult donors in response to peptide pools. 2×10⁵ PBMC/wellderived from six healthy adult donors were cultured with pools of two Fcpeptides (Table 3, see FIG. 13) for 4 days in the absence of exogenouslymphokines. IL-10 secretion by nTreg in response to peptide stimulationserved as a read out in these experiments and was measured in culturesupernatants by ELISA on day 4.

FIG. 4 of Example 3 (or FIG. 17):

Pools 3 and 4 are more immunogenic in healthy donors than in KDpatients. Panel A: IL-10 secretion in response to amino acid residues21-35 and 26-40 (pool 3) and 31-45 and 36-50 (pool 4) in healthy donors.Panel B: nTreg responses to pools 3 and 4 in KD patients: only 2 of 12KD subjects responded to these peptide pools.

FIG. 5 of Example 3 (or FIG. 18):

CD4+ CD25^(high) nTreg expansion in response to scalar doses of Fc. PBMCwere cultured for 4 days with 0, 1, 10, or 100 μg/ml purified Fcfragments. Panel A: Fc-specific nTreg response in adult subjects who hadKD in childhood. Panel B: Fc-specific nTreg response in healthy adultcontrols.

FIG. 6 of Example 3 (or FIG. 19):

HLA binding predictions of peptides derived from the Fc sequence. IEDBconsensus algorithm (www.iedb.org <http://www.iedb.org>) was used topredict HLA class II binding affinity of the Fc sequences described inTable 3, see FIG. 13. Immunogenic peptide pools are indicated.

REFERENCES—EXAMPLE 2

-   1. Burns, et al. 2004. Kawasaki syndrome. Lancet 364:533-544.-   2. Franco, et al. 2010. Memory T cells and characterization of    peripheral T cell clones in acute Kawasaki disease. Autoimmunity    43:317-324.-   3. Maddur, et al. 2010. Immunomodulation by intravenous    immunoglobulin: role of regulatory T cells. J Clin Immunol. 1:S4-8.-   4. Burns, et al. 2013. Immune-monitoring in Kawasaki disease    patients treated with infliximab and intravenous immunoglobulin.    Clinical and Experimental Immunol in press.-   5. Campos Ramos, et al. 2012. The autoimmune nature of post-infarct    myocardial healing: oral tolerance to cardiac antigens as a novel    strategy to improve cardiac healing. Autoimmunity 45:233-244.-   6. Trinath, et al. 2013. Intravenous immunoglobulin expands    regulatory T cells via induction of cyclooxygenase-2-dependent    prostaglandin E2 in human dendritic cells.

Blood in press.

-   7. Roncarolo, et al. 2007. Regulatory T cell immunotherapy for    tolerance to self antigens and to alloantigens in humans. Nature    Reviews 7:585-598.-   8. Wing, et al. 2010. Regulatory T cells exert checks and balances    on self tolerance and autoimmunity. Nature Immunology 11:7-13.-   9. von Boehmer, et al. 2010. Checkpoints in lymphocytes development    and autoimmune disease. Nature Immunology 11:14-20.-   10. Kassiotis, et al. 2008. Immunology. Immunity benefits from a    little suppression. Science 320:1168-1169.-   11. O'Garra, et al. 2004. IL-10-producing and naturally occurring    CD4+ Tregs: limiting collateral damage. J Clin Invest. 2004    114:1372-1378.-   12. Feuerer, et al. 2010. Foxp3+ regulatory T cells:    differentiation, specification, subphenotypes. Nature Immunology    10:698-695.-   13. Jordan, et al. 2001. Thymic selection of CD4+ CD25+ regulatory T    cells induced by an agonist self-peptide. Nat. Immunol. 2:301-306.-   14. Miyara, et al. 2009. Functional delineation and differentiation    dinamics of human CD4+ T cells expressing the FoxP3 transcription    factor. Immunity 30:899-911.-   15. Chen, et al. 2003. Conversion of Peripheral CD4+ CD25− Naive T    Cells to CD4+ CD25+ Regulatory T Cells by TGF-ß Induction of    Transcription Factor Foxp3. J. Exp. Med. 198:1875-1886.-   16. Kretschmer, et al. 2005. Inducing and expanding regulatory T    cell populations by foreign antigen. Nature Immunol. 6:1-9.-   17. Apostolou, et al. 2002. Origin of regulatory T cells with known    specificity for antigen. Nat. Immunol. 3:756-763.-   18. Apostolou, et al. 2004. In vivo instruction od suppressor    committment in naive T cells. Journal of Experimental Medicine    199:1401-1408.-   19. Rivino, et al. 2010. CCR6 is expressed on IL-10 producing,    autoreactive memory T cell population with context-dependent    regulatory function. J. Exp. Med. 207:565-577.-   20. Ohkura, et al. 2013. Development and mantainance of regulatory T    cells. Immunity 38:414-423.-   21. Chaudry, et al. 2013. Control of inflammation by integration of    environmental cues by regulatory T cells. J. Clin. Invest.    123:939-944.-   22. Sakaguchi, et al. 2013. The plasticity and stability of    regulatory T cells. Nature Review in Immunology 13:461-467.-   23. Povoleri, et al. 2013. Thymic versus induced regulatory T    cells—who regulates the regulators? Front Immunol. 2013 Jun. 27;    4:169. doi: 10.3389/fimmu.2013.00169. Print 2013. 4:169-   24. De Groot, et al. 2008. Activation of natural regulatory T cells    by IG Fc-derived peptide “Tregitopes”. Blood 112:3303-3311-   25. Franco, et al. 1992. Transferrin receptor mediates uptake and    presentation of hepatitis B envelope antigen by T lymphocytes J.    Exp. Med. 175:1195-1205.-   26. Zlotnik, et al. 2012. The chemokine superfamily revisited.    Immunity 36:705-716.-   27. Maddur, et al. 2011. Comparison of different IVIG preparations    on IL-17 production by human Th17 cells. Autoimmun. Rev. 12:809-810.-   28. Ephrem, et al. 2008. Expansion of CD4+ CD25+ regulatory T cells    by intravenous immunoglobulin: a critical factor in controlling    experimental autoimmune encephalitis. Blood 111:715-722.-   29. Reboldi, A et al. 2009. C—C chemokine receptor 6-regulated entry    of TH-17 cells into the CNS through the choroid plexus is required    for the initiation of EAE. Nature immunol. 10:514-523.-   30. Zielinski, et al. 2012. Pathogen-induced human TH17 cells    produce IFN-γ or IL-10 and are regulated by IL-1β. Nature    484:514-518.-   31. Ward, E. S. 2004. Acquiring maternal immunoglobulin; different    receptors, similar functions. Immunity 20:507-508.-   32. He, et al. 2008. FcRn-mediated antibody transport across    epithelial cells revealed by electron tomography. Nature    455:542-546.-   33. Rizzi, et al. 2005. In utero DNA immunisation. Immunity over    tolerance in fetal life. Vaccine 23:4273-4283.-   34. Bayry, et al. 2011. Intravenous immunoglobulin therapy in    rheumatic diseases. Nat Rev Rheumatol. 7:349-359.

REFERENCES—EXAMPLE 3

-   1. Jordan, et al. 2001. Thymic selection of CD4+ CD25+ regulatory T    cells induced by an agonist self-peptide. Nat. Immunol. 2:301-306.-   2. Miyara, et al. 2009. Functional delineation and differentiation    dinamic of human CD4+ T cells expressing the FoxP3 transcription    factor. Immunity 30:899-911.-   3. Franco, et al. 2014. Specificity of regulatory T cells that    modulate vascular inflammation. Autoimmunity 47:95-104.-   4. Ogata, et al. 2013. Treatment response in Kawasaki disease is    associated with sialylation levels of endogenous but not therapeutic    intravenous immunoglobulin G. PLoS one 8:e81448.-   5. Franco, A., Shimizu, C., Tremoulet, A. H., Burns, J. C. 2010.    Memory T cells and characterization of peripheral T cell clones in    acute Kawasaki disease. Autoimmunity 43:317-324.-   6. Franco, et al. 2010. Endoplasmic reticulum stress drives a    regulatory phenotype in human T cell clones. Cell. Immunol. 266:1-6.-   7. Burns, et al. 2013. Immune-monitoring in Kawasaki disease    patients treated with infliximab and intravenous immunoglobulin.    Clinical and Experimental Immunology 174:337-344.-   8. Wang, et al. 2010. Peptide binding predictions for HLA DR, DP and    DQ molecules. BMC Bioinformatics 11:568.-   9. Tangri, et al. 2005. Rationally engineered therapeutic proteins    with reduced immunogenicity. J Immunol. 174:3187-3196.-   10. Oseroff, et al. 2010. Molecular determinants of T cell epitope    recognition to the common Timothy grass allergen. Journal of    Immunology 185:943-955.-   11. Oseroff, et al. 2012. T Cell Responses to Known Allergen    Proteins Are Differently Polarized and Account for a Variable    Fraction of Total Response to Allergen Extracts. J. Immunol.    189:1800-1811.-   12. Lindestam Arlehamn, et al. 2013. Memory T cells in latent    Mycobacterium tuberculosis infection are directed against three    antigenic islands and largely contained in a CXCR3+CCR6+ Th1 subset.    PLoS Pathog. 9(1):e1003130. doi:    1003110.1001371/journal.ppat.1003130.-   13. Alexander, et al. 1994. Development of high potency universal    DR-restricted helper epitopes by modification of high affinity    DR-blocking peptides. Immunity 1:751-761.-   14. Paul, et al. 2013. Evaluating the immunogenicity of protein    drugs by applying in vitro MHC binding data and the immune epitope    database and analysis resource. 2013:467852. doi:    467810.461155/462013/467852.-   15. Arens, et al. 2008. Cutting edge: murine cytomegalovirus induces    a polyfunctional CD4 T cell response. J Immunol 180.-   16. Burns, J. C., Glode, M. P. 2004. Kawasaki syndrome. Lancet    364:533-544.-   17. De Groot, et al. 2008. Activation of natural regulatory T cells    by IG Fc-derived peptide “Tregitopes”. Blood 112:3303-3311.

A number of aspects of embodiments as provided herein have beendescribed. Nevertheless, it will be understood that variousmodifications may be made without departing from the spirit and scope ofthe invention. Accordingly, other aspects are within the scope of thefollowing claims.

1-18. (canceled) 19: A therapeutic composition comprising: a combinationof isolated, synthetic or recombinant peptides or polypeptidescomprising: (SEQ ID NO: 1) TAALGCLVKDYFPEP; (SEQ ID NO: 9)TFPAVLQSSGLYSLS; (SEQ ID NO: 11) LYSLSSVVTVPSSSL; (SEQ ID NO: 14)TYRVVSVLTVLHQDW; (SEQ ID NO: 45) SVLTVLHQDWLNGKE; (SEQ ID NO: 8)NNYKTTPPVLDSDGS; (SEQ ID NO: 16) QGNVFSCSVMHEALH; and (SEQ ID NO: 17)SCSVMHEALHNHYTQ,

20: The therapeutic composition of claim 19, wherein the combination ofisolated, synthetic or recombinant peptides or polypeptides isformulated for administration: via an enteral or a parenteral route, ororally, subcutaneously, or systemically or topically; via a parenteralroute comprising a subcutaneous, an intravenous (IV), an intradermal, anintramuscular, an intraperitoneal, an intranasal, a transdermal or abuccal route; intradermally as a sterile formulation; or as a controlledrelease formulation, or formulated in a tablet, a pill, a gel, a patch,in an implant or in a spray, as an inhaled powder. 21: The therapeuticcomposition of claim 19, wherein the combination of isolated, syntheticor recombinant peptides or polypeptides is formulated foradministration: parenterally by one bolus injection, or by two, three orfour or more bolus injections, or by gradual perfusion over time; atintervals of 1 week, 2 weeks, 4 weeks (or one month), 6 weeks, 8 weeksor one year; or at a daily dose of peptide in a range of about 10nanograms to 10 milligrams, or about 1 microgram to 10 milligrams.22-25. (canceled) 26: The therapeutic composition of claim 19, whereinany one of the isolated, synthetic or recombinant peptide or polypeptideof the combination of isolated, synthetic or recombinant peptides orpolypeptides has at least one conservative amino acid substitution andretains its property of generating natural regulatory T cells (nTregs)that can suppress pro-inflammatory T cells and pro-inflammatory T cellresponses when administered to an individual. 27: The therapeuticcomposition of claim 26, wherein the at least one conservative aminoacid substitution comprises: substituting an amino acid with anotheramino acid of like characteristics; or, a replacement of an aliphaticamino acid with another aliphatic amino acid; a replacement of a Serinewith a Threonine or vice versa; a replacement of an acidic residue withanother acidic residue; a replacement of a residue bearing an amidegroup with another residue bearing an amide group; an exchange of abasic residue with another basic residue: or a replacement of anaromatic residue with another aromatic residue, and optionally thepeptide or polypeptide, or at least one amino acid in the peptide orpolypeptide, comprises or consists of a peptidomimetic or a non-naturalamino acid residue. 28: The therapeutic composition of claim 19, whereinany one of the isolated, synthetic or recombinant peptides orpolypeptides of the combination of isolated, synthetic or recombinantpeptides or polypeptides is formulated as a pharmaceutical compositionor a formulation, and optionally the pharmaceutical composition orformulation comprises a pharmaceutically acceptable excipient. 29: Thetherapeutic composition of claim 19, wherein any one of the isolated,synthetic or recombinant peptides or polypeptides of the combination ofisolated, synthetic or recombinant peptides or polypeptides isformulated in a sterile solution for injection, or as a powder or alyophilized or a freeze-dried composition. 30: The therapeuticcomposition of claim 19, wherein any one of the polypeptide or peptidesor polypeptides of the combination of isolated, synthetic or recombinantpeptides or polypeptides is formulated in or as a liposome or ananoparticle. 31: The therapeutic composition of claim 19, wherein anyone of the polypeptide or peptides or polypeptides of the combination ofisolated, synthetic or recombinant peptides or polypeptides isformulated in or as a vaccine. 32-33. (canceled) 34: The therapeuticcomposition of claim 19, wherein the combination of isolated, syntheticor recombinant peptides or polypeptides consists of: (SEQ ID NO: 1)TAALGCLVKDYFPEP; (SEQ ID NO: 9) TFPAVLQSSGLYSLS; (SEQ ID NO: 11)LYSLSSVVTVPSSSL; (SEQ ID NO: 14) TYRVVSVLTVLHQDW; (SEQ ID NO: 45)SVLTVLHQDWLNGKE; (SEQ ID NO: 8) NNYKTTPPVLDSDGS; (SEQ ID NO: 16)QGNVFSCSVMHEALH; and, (SEQ ID NO: 17) SCSVMHEALHNHYTQ.

35: The therapeutic composition of claim 19, wherein the combination ofisolated, synthetic or recombinant peptides or polypeptides isformulated for administration as a vaccine with an adjuvant. 36: Thetherapeutic composition of claim 19, wherein the combination ofisolated, synthetic or recombinant peptides or polypeptides consistsessentially of: (SEQ ID NO: 1) TAALGCLVKDYFPEP; (SEQ ID NO: 9)TFPAVLQSSGLYSLS; (SEQ ID NO: 11) LYSLSSVVTVPSSSL; (SEQ ID NO. 14)TYRVVSVLTVLHQDW; (SEQ ID NO: 45) SVLTVLHQDWLNGKE; (SEQ ID NO: 8)NNYKTTPPVLDSDGS; (SEQ ID NO: 16) QGNVFSCSVMHEALH; and, (SEQ ID NO: 17)SCSVMHEALHNHYTQ.